Title page. Negative effect of clenbuterol on physical capacities and neuromuscular control of muscle atrophy in adult rats. Authors: Guillaume Lang (MSc.1), Valérie Dernoncourt (MSc.1), Jean-François Bisson (PhD.1).
Affiliations:
1
ETAP-Ethologie Appliquée, 13 rue du Bois de la Champelle, 54500,
Vandœuvre-lès-Nancy, France.
Acknowledgments: The authors wish to thank Ms. D.M. Wood for proofing the English language in this manuscript.
Name and postal and email addresses for the corresponding author: Guillaume Lang. ETAP-Ethologie Appliquée, 13 rue du Bois de la Champelle, 54500, Vandœuvre-lès-Nancy, France. [email protected]
Running title: Muscle mass and physical capacities.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24273
Muscle & Nerve
Page 2 of 33 Muscle mass and physical capacities. 2
Abstract. Introduction. Clenbuterol has been used to alleviate chronic obstructive pulmonary disease and elicit an anabolic response in muscles. The aim of this project was to evaluate the influence of muscle mass variation on physical capacities in rats. Methods. The left hindlimbs of Wistar rats were immobilized for 20 days in plantar flexion with a splint and then remobilized for 16 days. The effect of a non-myotoxic dose of clenbuterol during the immobilization period was evaluated. Physical capacities were coordination, free locomotion, grip strength, and bilateral deficit. Results. Immobilization induced a loss of muscle mass, coordination, and strength without any effect on free locomotion. The positive anabolic effect of clenbuterol did not prevent a loss of physical capacities resulting from immobilization. Discussion. Muscle mass was correlated strongly with coordination and isometric strength in untreated rats. Anabolic effect, fiber phenotype modification, and perturbation in neuromuscular communication with clenbuterol could improve muscle mass, but it altered physical capacities.
Key words. Muscle mass, locomotion, coordination, strength, clenbuterol.
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Page 3 of 33
Muscle & Nerve Muscle mass and physical capacities. 3
Abbreviation list. BIA: Bioimpedance analysis. BLD: Bilateral deficit. BW: Body weight. CELMEA: Comité d’Ethique Lorrain en Matière d'Expérimentation Animale. COPD: Chronic obstructive pulmonary disease. CSA: Cross sectional area. CT: Computed tomography. D: Day. DXA: Dual energy X-ray absorptiometry. EDL: Extensor digitorum longus. EWGSOP: European Working Group on Sarcopenia in Older People. GA: Gastrocnemius. I.P.: Intraperitoneal. MRI: Magnetic resonance imaging. MVC: Maximum volontary contraction. N: Newton. PL: Plantaris. SARMs: Selective androgen receptor modulators. S.C.: Subcutaneous. SOL: Soleus. SPPB: Short physical performance battery. TA: Tibialis anterior.
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Muscle & Nerve
Page 4 of 33 Muscle mass and physical capacities. 4
Introduction. Muscle atrophy occurs in various conditions, such as cancer, AIDS, chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF), chronic renal failure, aging, loss of activity, and immobilization following a fracture or injury (1,2). A loss of muscle mass is often associated with a loss of physical abilities in patients. For example, immobilization following a fracture requires rehabilitation for good motor capacity recovery (3). Aging induces spontaneous loss of muscle mass, which increases the risk of fall and a loss of independence (4). There is little information however, on the evolution of physical capacities during muscle atrophy and recovery. In 2010, the European Working Group on Sarcopenia in Older People (EWGSOP) recommended using the presence of both low muscle mass and low physical capacities (strength or performance) for the diagnosis of sarcopenia (5). Body imaging techniques such as computed tomography (CT scan), magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DXA), are used to estimate muscle mass or lean body mass, and bioimpedance analysis (BIA) is used to measure fat volume and lean body mass. Coordination and free locomotion are measured with the Short Physical Performance Battery (SPPB), which includes usual gait speed, a 6 minute walking test, and the stair climb power test (5, 6). Muscle strength is evaluated by tests of handgrip, knee flexion/extension, and peak expiratory flow. Bilateral and unilateral strengths are used to detect the neuromuscular control deviation called the bilateral deficit (BLD) (7, 8). Models of muscle atrophy differ by their mode of induction (for example, tenotomy, denervation, tetanus toxin, undernutrition, or immobilization), duration, intensity, reversibility, and atrophied muscles (9, 10). The advantage of the immobilization model
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Page 5 of 33
Muscle & Nerve Muscle mass and physical capacities. 5
is to quickly achieve atrophy in 1 hindlimb with recovery after remobilization. A number of articles have shown that hindlimb immobilization induces significant muscle atrophy with changes in the ultrastructure of collagen (11), increased muscle tension, and the quantity of connective tissue (12). It decreases muscle density, muscle cross sectional area (CSA) (13, 14), fiber diameter (11), the number of muscle fiber nuclei, and the location and number of sarcomeres (15). Biological analysis has reported an imbalance between muscle protein synthesis and break-down during immobilization (16; 17). These modifications are related to the position and duration of muscle immobilization. We used this model of muscle atrophy in rats to study the link between loss and recovery of muscle mass and physical capacities. Inspired by tests in patients, we evaluated physical capacities by coordination, free locomotion, and isometric strength in rats. The first aim of this study was to evaluate the effect of immobilization and remobilization on muscle mass, physical capacities, and neuromuscular control. The second aim was to study the effect of the anabolic drug clenbuterol on muscle mass, physical capacities, and neuromuscular control in immobilized rats.
Materials and Methods. Animals and Ethical approval. Seventy-two male Wistar rats (Charles River, L'Arbresle, France) of 300-400 g and 9 weeks old were used and housed in a reversed light cycle (light from 8 pm to 8 am). Animals were maintained under conditions of controlled temperature (22±2°C) and humidity (50±20%). They had free access to a standard diet (2016, Teklab diet, Harlan, Indianapolis, Indiana, U.S.A.) and drinking water ad libitum. Animals housed were 2 per cage. All procedures were conducted in
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Muscle & Nerve
Page 6 of 33 Muscle mass and physical capacities. 6
conformity with the institutional guidelines and ethics committee: CELMEA number 66, agreement no.00041-01. Experimental groups. Seventy-two rats were divided into 3 groups (24 per group): the non-immobilized control group which did not have an immobilized left hindlimb; the immobilized-only group, which had an immobilized left hindlimb for 20 days (D0 to D20); and the immobilized and treated group, which had an immobilized left hindlimb and was treated with clenbuterol for 20 days (D0 to D20). In all groups, the right hindlimb was never immobilized. Each group was separated into 3 equal subgroups (8 per sub-group): no remobilization (sacrificed on D20); 1 week of remobilization (sacrificed on D28); or 2 weeks (sacrificed on D36) of remobilization. Induction of muscle atrophy. Muscle atrophy was induced by left hindlimb immobilization with a splint made of aluminium and foam. The splint weight was 5.3 ± 0.1 g corresponding to approximately 2% of rat body weight at the beginning of the study. The left hindlimb was immobilized in plantar flexion for 20 days (D0 to D20). The right hindlimb was the contralateral intact paw. Immobilization was performed without anesthesia, as this device did not produce pain in rats. The splint was placed around the ankle joint, and the diameter was adjusted to reduce the risk of edema and inflammation. Animals were observed every 2 hours on D0 to assess their behavior and the appearance of the immobilized paw. From D1, the observations and diameter adjustments were made twice daily if necessary. At the end of the immobilization period, the splint was removed, and rats were able to move freely (remobilization period, D20 to D36). Drug administration. Clenbuterol hydrochloride was obtained from SigmaAldrich (Sigma-Aldrich Chemie, Saint-Quentin Fallavier, France) and solubilized in
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Page 7 of 33
Muscle & Nerve Muscle mass and physical capacities. 7
sodium chloride at 0.9% (C.D.M. Lavoisier, Paris, France). The solution of clenbuterol was injected at 5 µg/kg by the subcutaneous route, twice daily for 20 days in the immobilized and treated group. Physical capacities. The rotarod, open field, and grip strength tests were all performed before immobilization (D0), at the end of the immobilization period (D20), and once per week for 2 weeks of remobilization (D28 and D36). The temperature (22±1°C) and light intensity (35±3 lumens) of the test room, the position of the device, and the sequence of tests were similar throughout the study. The same persons performed these tests in order to avoid variability. By sub-group, physical capacities were measured in rats without (D20), with 1 week (D28), or with 2 weeks (D36) of remobilization on D0-D20, D0-D20-D28, and D0-D20-D28-D36, respectively. Coordination in rodents is commonly measured using a rotarod (18). The length of time that animals stay on this rotating rod is a measure of their balance, coordination, physical condition, and motor planning (19; 20; 21). Coordination was observed using a rotarod over 3 trials. Rats were acclimated to the rotarod 1 day before the first test (D-1) under the same conditions as the trial. Coordination was measured by the times on the cylinder with speed increases from 4 to 40 rpm for 5 minutes maximum without reversed rotation. Free locomotion is observed using an open-field arena (22). The open field test allows for evaluation of the quality and quantity of locomotion activity in rats. Parameters such as distance moved, time spent moving and rearing allows for the observation of general activity (23). Rats were placed into a Plexiglas® cube (Loraplast, Essey-lès-Nancy, France) with black walls (60 x 60 cm) and a floor that was divided into 9 virtually equal quadrants. Each rat was recorded for 5 minutes using a flexible video tracking system
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Muscle & Nerve
Page 8 of 33 Muscle mass and physical capacities. 8
(Any-maze®, Wood Dale, Illinois, U.S.A.). Free locomotion was measured by distance (m), number of lines crossed, and the number and duration (s) of rearings. Isometric strength can be measured by maximum grip strength (24) using a dynamometer (25-28). The grip strength test measured the maximal force peak (Newton, N) developed by each hindlimb (right or left hindlimb unilateral isometric strength) or both hindlimbs (bilateral isometric strength). The measurement was accomplished using a highly accurate dynamometer (Digital dynamometer FK, Sauter®, Balingen, Germany) with a sampling rate of 1000 Hz. The animal was held by the tail and under the forelimbs. Each hindlimb or both hindlimbs were in contact with the grid. The animal was slid along the axis of the sensor, and maximum voltage was recorded 3 times until the animal released the grid. Neuromuscular control. Neuromuscular control can be calculated from maximum grip strength (24). The neuromuscular control deviation, called bilateral deficit (BLD) is the difference between bilateral strength and summed unilateral strengths: BLD (%) = [(Sum of the unilateral strengths - Bilateral strength) ÷ Sum of the unilateral strengths] x 100 Evaluation of muscle atrophy. On D20 (sub-groups without remobilization), D28 (sub-groups with 1 week of remobilization), and D36 (sub-groups with 2 weeks of remobilization), 8 non-immobilized rats, 8 rats only immobilized, and 8 immobilized and treated rats were sacrificed to evaluate muscle mass. The hindlimb muscles (soleus: SOL, gastrocnemius: GA, plantaris: PL, tibialis anterior: TA, and extensor digitorum longus: EDL) were weighed after euthanasia by an overdose of sodium pentobarbital
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Page 9 of 33
Muscle & Nerve Muscle mass and physical capacities. 9
(CEVA®, Libourne, France, 200 mg/kg, i.p.: intraperitoneal route). Muscle atrophy was expressed in gram of muscle per kilogram of body weight (g/kg BW). Statistical analysis. One-way ANOVA with Bonferroni post-hoc correction were used to compare the mean and standard error (SEM) per group at each time. Statistical analyses were performed using StatView® 5 statistical package (SAS Institute, Cary, North Carolina, U.S.A.).
Results. Physical capacities (Figs. 1 to 3). Physical capacities were studied by 3 tests: coordination with a rotarod, free locomotion in an open field, and hindlimb isometric strength with the grip strength test. Before the immobilization period (D0), there was no significant difference between the groups in coordination, free locomotion, or isometric strength. Coordination with a rotarod (Fig. 1): At the end of the immobilization period (D20), the time on the cylinder decreased (-45%, P
Affiliations:
1
ETAP-Ethologie Appliquée, 13 rue du Bois de la Champelle, 54500,
Vandœuvre-lès-Nancy, France.
Acknowledgments: The authors wish to thank Ms. D.M. Wood for proofing the English language in this manuscript.
Name and postal and email addresses for the corresponding author: Guillaume Lang. ETAP-Ethologie Appliquée, 13 rue du Bois de la Champelle, 54500, Vandœuvre-lès-Nancy, France. [email protected]
Running title: Muscle mass and physical capacities.
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.24273
Muscle & Nerve
Page 2 of 33 Muscle mass and physical capacities. 2
Abstract. Introduction. Clenbuterol has been used to alleviate chronic obstructive pulmonary disease and elicit an anabolic response in muscles. The aim of this project was to evaluate the influence of muscle mass variation on physical capacities in rats. Methods. The left hindlimbs of Wistar rats were immobilized for 20 days in plantar flexion with a splint and then remobilized for 16 days. The effect of a non-myotoxic dose of clenbuterol during the immobilization period was evaluated. Physical capacities were coordination, free locomotion, grip strength, and bilateral deficit. Results. Immobilization induced a loss of muscle mass, coordination, and strength without any effect on free locomotion. The positive anabolic effect of clenbuterol did not prevent a loss of physical capacities resulting from immobilization. Discussion. Muscle mass was correlated strongly with coordination and isometric strength in untreated rats. Anabolic effect, fiber phenotype modification, and perturbation in neuromuscular communication with clenbuterol could improve muscle mass, but it altered physical capacities.
Key words. Muscle mass, locomotion, coordination, strength, clenbuterol.
John Wiley & Sons, Inc.
Page 3 of 33
Muscle & Nerve Muscle mass and physical capacities. 3
Abbreviation list. BIA: Bioimpedance analysis. BLD: Bilateral deficit. BW: Body weight. CELMEA: Comité d’Ethique Lorrain en Matière d'Expérimentation Animale. COPD: Chronic obstructive pulmonary disease. CSA: Cross sectional area. CT: Computed tomography. D: Day. DXA: Dual energy X-ray absorptiometry. EDL: Extensor digitorum longus. EWGSOP: European Working Group on Sarcopenia in Older People. GA: Gastrocnemius. I.P.: Intraperitoneal. MRI: Magnetic resonance imaging. MVC: Maximum volontary contraction. N: Newton. PL: Plantaris. SARMs: Selective androgen receptor modulators. S.C.: Subcutaneous. SOL: Soleus. SPPB: Short physical performance battery. TA: Tibialis anterior.
John Wiley & Sons, Inc.
Muscle & Nerve
Page 4 of 33 Muscle mass and physical capacities. 4
Introduction. Muscle atrophy occurs in various conditions, such as cancer, AIDS, chronic obstructive pulmonary disease (COPD), congestive heart failure (CHF), chronic renal failure, aging, loss of activity, and immobilization following a fracture or injury (1,2). A loss of muscle mass is often associated with a loss of physical abilities in patients. For example, immobilization following a fracture requires rehabilitation for good motor capacity recovery (3). Aging induces spontaneous loss of muscle mass, which increases the risk of fall and a loss of independence (4). There is little information however, on the evolution of physical capacities during muscle atrophy and recovery. In 2010, the European Working Group on Sarcopenia in Older People (EWGSOP) recommended using the presence of both low muscle mass and low physical capacities (strength or performance) for the diagnosis of sarcopenia (5). Body imaging techniques such as computed tomography (CT scan), magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DXA), are used to estimate muscle mass or lean body mass, and bioimpedance analysis (BIA) is used to measure fat volume and lean body mass. Coordination and free locomotion are measured with the Short Physical Performance Battery (SPPB), which includes usual gait speed, a 6 minute walking test, and the stair climb power test (5, 6). Muscle strength is evaluated by tests of handgrip, knee flexion/extension, and peak expiratory flow. Bilateral and unilateral strengths are used to detect the neuromuscular control deviation called the bilateral deficit (BLD) (7, 8). Models of muscle atrophy differ by their mode of induction (for example, tenotomy, denervation, tetanus toxin, undernutrition, or immobilization), duration, intensity, reversibility, and atrophied muscles (9, 10). The advantage of the immobilization model
John Wiley & Sons, Inc.
Page 5 of 33
Muscle & Nerve Muscle mass and physical capacities. 5
is to quickly achieve atrophy in 1 hindlimb with recovery after remobilization. A number of articles have shown that hindlimb immobilization induces significant muscle atrophy with changes in the ultrastructure of collagen (11), increased muscle tension, and the quantity of connective tissue (12). It decreases muscle density, muscle cross sectional area (CSA) (13, 14), fiber diameter (11), the number of muscle fiber nuclei, and the location and number of sarcomeres (15). Biological analysis has reported an imbalance between muscle protein synthesis and break-down during immobilization (16; 17). These modifications are related to the position and duration of muscle immobilization. We used this model of muscle atrophy in rats to study the link between loss and recovery of muscle mass and physical capacities. Inspired by tests in patients, we evaluated physical capacities by coordination, free locomotion, and isometric strength in rats. The first aim of this study was to evaluate the effect of immobilization and remobilization on muscle mass, physical capacities, and neuromuscular control. The second aim was to study the effect of the anabolic drug clenbuterol on muscle mass, physical capacities, and neuromuscular control in immobilized rats.
Materials and Methods. Animals and Ethical approval. Seventy-two male Wistar rats (Charles River, L'Arbresle, France) of 300-400 g and 9 weeks old were used and housed in a reversed light cycle (light from 8 pm to 8 am). Animals were maintained under conditions of controlled temperature (22±2°C) and humidity (50±20%). They had free access to a standard diet (2016, Teklab diet, Harlan, Indianapolis, Indiana, U.S.A.) and drinking water ad libitum. Animals housed were 2 per cage. All procedures were conducted in
John Wiley & Sons, Inc.
Muscle & Nerve
Page 6 of 33 Muscle mass and physical capacities. 6
conformity with the institutional guidelines and ethics committee: CELMEA number 66, agreement no.00041-01. Experimental groups. Seventy-two rats were divided into 3 groups (24 per group): the non-immobilized control group which did not have an immobilized left hindlimb; the immobilized-only group, which had an immobilized left hindlimb for 20 days (D0 to D20); and the immobilized and treated group, which had an immobilized left hindlimb and was treated with clenbuterol for 20 days (D0 to D20). In all groups, the right hindlimb was never immobilized. Each group was separated into 3 equal subgroups (8 per sub-group): no remobilization (sacrificed on D20); 1 week of remobilization (sacrificed on D28); or 2 weeks (sacrificed on D36) of remobilization. Induction of muscle atrophy. Muscle atrophy was induced by left hindlimb immobilization with a splint made of aluminium and foam. The splint weight was 5.3 ± 0.1 g corresponding to approximately 2% of rat body weight at the beginning of the study. The left hindlimb was immobilized in plantar flexion for 20 days (D0 to D20). The right hindlimb was the contralateral intact paw. Immobilization was performed without anesthesia, as this device did not produce pain in rats. The splint was placed around the ankle joint, and the diameter was adjusted to reduce the risk of edema and inflammation. Animals were observed every 2 hours on D0 to assess their behavior and the appearance of the immobilized paw. From D1, the observations and diameter adjustments were made twice daily if necessary. At the end of the immobilization period, the splint was removed, and rats were able to move freely (remobilization period, D20 to D36). Drug administration. Clenbuterol hydrochloride was obtained from SigmaAldrich (Sigma-Aldrich Chemie, Saint-Quentin Fallavier, France) and solubilized in
John Wiley & Sons, Inc.
Page 7 of 33
Muscle & Nerve Muscle mass and physical capacities. 7
sodium chloride at 0.9% (C.D.M. Lavoisier, Paris, France). The solution of clenbuterol was injected at 5 µg/kg by the subcutaneous route, twice daily for 20 days in the immobilized and treated group. Physical capacities. The rotarod, open field, and grip strength tests were all performed before immobilization (D0), at the end of the immobilization period (D20), and once per week for 2 weeks of remobilization (D28 and D36). The temperature (22±1°C) and light intensity (35±3 lumens) of the test room, the position of the device, and the sequence of tests were similar throughout the study. The same persons performed these tests in order to avoid variability. By sub-group, physical capacities were measured in rats without (D20), with 1 week (D28), or with 2 weeks (D36) of remobilization on D0-D20, D0-D20-D28, and D0-D20-D28-D36, respectively. Coordination in rodents is commonly measured using a rotarod (18). The length of time that animals stay on this rotating rod is a measure of their balance, coordination, physical condition, and motor planning (19; 20; 21). Coordination was observed using a rotarod over 3 trials. Rats were acclimated to the rotarod 1 day before the first test (D-1) under the same conditions as the trial. Coordination was measured by the times on the cylinder with speed increases from 4 to 40 rpm for 5 minutes maximum without reversed rotation. Free locomotion is observed using an open-field arena (22). The open field test allows for evaluation of the quality and quantity of locomotion activity in rats. Parameters such as distance moved, time spent moving and rearing allows for the observation of general activity (23). Rats were placed into a Plexiglas® cube (Loraplast, Essey-lès-Nancy, France) with black walls (60 x 60 cm) and a floor that was divided into 9 virtually equal quadrants. Each rat was recorded for 5 minutes using a flexible video tracking system
John Wiley & Sons, Inc.
Muscle & Nerve
Page 8 of 33 Muscle mass and physical capacities. 8
(Any-maze®, Wood Dale, Illinois, U.S.A.). Free locomotion was measured by distance (m), number of lines crossed, and the number and duration (s) of rearings. Isometric strength can be measured by maximum grip strength (24) using a dynamometer (25-28). The grip strength test measured the maximal force peak (Newton, N) developed by each hindlimb (right or left hindlimb unilateral isometric strength) or both hindlimbs (bilateral isometric strength). The measurement was accomplished using a highly accurate dynamometer (Digital dynamometer FK, Sauter®, Balingen, Germany) with a sampling rate of 1000 Hz. The animal was held by the tail and under the forelimbs. Each hindlimb or both hindlimbs were in contact with the grid. The animal was slid along the axis of the sensor, and maximum voltage was recorded 3 times until the animal released the grid. Neuromuscular control. Neuromuscular control can be calculated from maximum grip strength (24). The neuromuscular control deviation, called bilateral deficit (BLD) is the difference between bilateral strength and summed unilateral strengths: BLD (%) = [(Sum of the unilateral strengths - Bilateral strength) ÷ Sum of the unilateral strengths] x 100 Evaluation of muscle atrophy. On D20 (sub-groups without remobilization), D28 (sub-groups with 1 week of remobilization), and D36 (sub-groups with 2 weeks of remobilization), 8 non-immobilized rats, 8 rats only immobilized, and 8 immobilized and treated rats were sacrificed to evaluate muscle mass. The hindlimb muscles (soleus: SOL, gastrocnemius: GA, plantaris: PL, tibialis anterior: TA, and extensor digitorum longus: EDL) were weighed after euthanasia by an overdose of sodium pentobarbital
John Wiley & Sons, Inc.
Page 9 of 33
Muscle & Nerve Muscle mass and physical capacities. 9
(CEVA®, Libourne, France, 200 mg/kg, i.p.: intraperitoneal route). Muscle atrophy was expressed in gram of muscle per kilogram of body weight (g/kg BW). Statistical analysis. One-way ANOVA with Bonferroni post-hoc correction were used to compare the mean and standard error (SEM) per group at each time. Statistical analyses were performed using StatView® 5 statistical package (SAS Institute, Cary, North Carolina, U.S.A.).
Results. Physical capacities (Figs. 1 to 3). Physical capacities were studied by 3 tests: coordination with a rotarod, free locomotion in an open field, and hindlimb isometric strength with the grip strength test. Before the immobilization period (D0), there was no significant difference between the groups in coordination, free locomotion, or isometric strength. Coordination with a rotarod (Fig. 1): At the end of the immobilization period (D20), the time on the cylinder decreased (-45%, P
