Elsevier

Bone

Volume 46, Issue 1, January 2010, Pages 24-31
Bone

Muscle and bone follow similar temporal patterns of recovery from muscle-induced disuse due to botulinum toxin injection

https://doi.org/10.1016/j.bone.2009.10.016Get rights and content

Abstract

If muscle force is a primary source for triggering bone adaptation, with disuse and reloading, bone changes should follow muscle changes. We examined the timing and magnitude of changes in muscle cross-sectional area (MCSA) and bone architecture in response to muscle inactivity following botulinum toxin (BTX) injection. We hypothesized that MCSA would return to baseline levels sooner than bone properties following BTX injection.

Female BALB mice (15 weeks old) were injected with 20 μL of BTX (1 U/100 g body mass, n = 18) or saline (SAL, n = 18) into the posterior calf musculature of one limb. The contralateral limb (CON) served as an internal control. MCSA and bone properties were assessed at baseline, 2, 4, 8, 12, and 16 weeks post-injection using in vivo micro-CT at the tibia proximal metaphysis (bone only) and diaphysis. Muscles were dissected and weighed after sacrifice.

Significant Group × Leg × Time interactions indicated that the maximal decrease in MCSA (56%), proximal metaphyseal BV/TV (38%) and proximal diaphyseal Ct.Ar (7%) occurred 4 weeks after injection. There was no delay prior to bone recovery as both muscle and bone properties began to recover after this time, but MCSA and BV/TV remained 15% and 20% lower, respectively, in the BTX-injected leg than the BTX-CON leg 16 weeks post-injection. Gastrocnemius mass (primarily fast-twitch) was 14% lower in the BTX-injected leg than the SAL-injected leg, while soleus mass (primarily slow-twitch) was 15% greater in the BTX group than the SAL group.

Our finding that muscle size and bone began to recover at similar times after BTX injection was unexpected. This suggested that partial weight-bearing and/or return of slow-twitch muscle activity in the BTX leg may have been sufficient to stimulate bone recovery. Alternatively, muscle function may have recovered sooner than MCSA. Our results indicated that muscle cross-sectional area, while important, may not be the primary factor associated with bone loss and recovery when muscle atrophy is induced through BTX injection. To understand the nature of the interaction between muscle and bone, future work should focus on the functional recovery of individual muscles in relation to bone.

Introduction

Bone and muscle, ostensibly, grow in proportion to one another [1]. This is explained biomechanically by the observation that muscles impose large loads on bone. Indeed, on a population level, when grouped by sex and maturity level (e.g., pre-pubertal, post-pubertal, and menopausal), muscle and bone mass are highly correlated [2], [3]. However there are also many examples, such as the myostatin-null mouse [4], where genetics or other factors may override the biomechanical link between muscle and bone [5]. Burr [6] suggested that if muscle force is the primary source for triggering bone adaptation, in a disuse situation, a decrease in muscle strength must precede a decrease in bone strength. Conversely, when reloaded, an increase in muscle strength must precede an increase in bone strength.

There are many factors that determine the rate of atrophy and the ability to recover following disuse. In bone, recovery from disuse takes longer than bone loss [7], [8]. Part of the temporal lag in bone recovery is associated with the relatively longer formation period following the resorption period [9], [10], as well as the delay between new bone formation and full mineralization of the new bone [6]. In addition, after disuse there is a decrease in the number of trabeculae resulting in reduced bone surface available for bone formation [11]. In muscle, satellite cells are activated when muscle growth or regeneration is required after atrophy [12]. The satellite cells fuse to damaged myoibers and produce new myofibers. In addition, younger animals are more likely to regain pre-disuse levels of bone and muscle than older animals [13], [14]. Although studies of individual tissues provide valuable insight into the capacity of each tissue to recover from disuse, it is difficult to compare among species, ages, and means of achieving disuse (e.g., immobilization with a cast or hindlimb unloading).

Few studies have investigated concomitant longitudinal changes in muscle and bone. A single study in rats that examined both muscle and bone during recovery from hindlimb unloading found that both muscle mass and muscle torque production return to normal levels sooner than bone mineral content and density [8]. Secondly, in an ACL-injured patient examined pre- and post-injury, knee extensor strength decreased immediately following the injury, while the maximal decrease in patellar bone mineral density was delayed by several weeks [15]. Muscle strength began to increase 5 weeks post-injury, while bone mineral density began to increase only 14 weeks post-injury. Other than this single case-study, knowledge of the timeline for regaining bone strength and muscle strength is based on models where both weight-bearing and muscle activity are affected.

In addition to muscle-induced loads, significant loads are also imparted on the skeleton through weight-bearing. Intra-muscular injection of botulinum toxin type A (BTX) in a murine model can be an effective means of eliciting muscular disuse with minimal effects on gait [16]. This muscular disuse led to bone loss [16], [17], [18]. Thus, unlike hindlimb unloading, some weight-bearing may be permitted in the affected limb following BTX injection, while muscle activity is impaired for several weeks. Muscular recovery from BTX injection differs from other patterns of disuse because new motor nerve terminals must be formed to create new neuromuscular connections [19], [20], [21]. In rodent models where relatively high doses of BTX are injected, recovery to normal muscle size has been reported to take 8–18 weeks [19], [21].

The aim of this study was to follow the concomitant timing and magnitude of changes in muscle cross-sectional area and bone microarchitecture that occur in response to muscle inactivity following BTX injection to the posterior hindlimb of the mouse. We hypothesized that muscle cross-sectional area would return to baseline levels sooner than bone properties following BTX injection. In addition, we examined how bone microarchitecture adapted to the absence and regain of muscle activity.

Section snippets

Animal model and experimental design

Thirty-six skeletally mature (15 weeks old) [22], [23] BALB/cAnNCrl (BALB) female mice were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada). They were randomly assigned to one of two groups. Based on group assignment, they were injected with 20 μL of (1) BTX Type A (BOTOX, Allergan, Inc., 1 U/100 g body weight) or (2) saline (SAL) into the posterior lower limb musculature (a single injection targeting the gastrocnemius, plantaris, and soleus). Within each group, mice

Body mass

At the beginning of the study, the mice weighed a mean (± SD) of 20.6 ± 0.2 g. Although body mass appeared to decrease in the BTX group following injection (Fig. 1), there was no Group main effect (p = 0.10) or Group × Time interaction (p = 0.24).

Muscle properties

There was a significant Group × Leg × Time interaction for MCSA (p < 0.001) (Fig. 1). MCSA decreased in the BTX-INJ leg 2 weeks following injection, and remained 15% lower than the contralateral control leg (BTX-CON) and 20% lower than the SAL-INJ leg at W16 (Fig. 2

Discussion

Muscle imposes significant loads on bone, which can drive bone adaptation. In this study of skeletally mature BALB mice, we found that contrary to our hypothesis, muscle and bone properties recovered at similar times following BTX injection. Our results showed that MCSA, BV/TV, and Ct.Ar decreased maximally 4 weeks post-injection and began to recover thereafter. These results suggested that MCSA may not be the primary factor associated with bone recovery following BTX injection. The significant

Acknowledgments

The authors thank Helen Buie for assistance with micro-CT image analysis and assistance with the autosegmentation protocol, as well as Tim Leonard, Dr. Walter Herzog, and Dr. Ted Gross for advice regarding BTX injection. We also acknowledge Dr. Tak Fung for assistance with statistical analyses. This study was supported, in part, by the University of Michigan Department of Orthopaedic Surgery. Sarah L. Manske was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC)

References (45)

  • Y.K. Luu et al.

    In vivo quantification of subcutaneous and visceral adiposity by micro-computed tomography in a small animal model

    Med. Eng. Phys.

    (2009)
  • M. Sode et al.

    Resolution dependence of the non-metric trabecular structure indices

    Bone

    (2008)
  • H.R. Buie et al.

    Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis

    Bone

    (2007)
  • A. Odgaard et al.

    Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions

    Bone

    (1993)
  • B.F. Boyce et al.

    Required and nonessential functions of nuclear factor-kappa B in bone cells

    Bone

    (1999)
  • M. Adler et al.

    Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle

    Toxicon

    (2001)
  • W. Herzog et al.

    The role of muscles in joint degeneration and osteoarthritis

    J. Biomech.

    (2007)
  • M. Yaraskavitch et al.

    Botox produces functional weakness in non-injected muscles adjacent to the target muscle

    J. Biomech.

    (2008)
  • D.W. Thompson

    On growth and form (abridged edition)

    (1961)
  • D.B. Burr

    Muscle strength, bone mass, and age-related bone loss

    J. Bone Miner. Res.

    (1997)
  • M.H. Lafage-Proust et al.

    Space-related bone mineral redistribution and lack of bone mass recovery after reambulation in young rats

    Am. J. Physiol.

    (1998)
  • M.R. Allen et al.

    Differential bone and muscle recovery following hindlimb unloading in skeletally mature male rats

    J. Musculoskelet. Neuronal Interact.

    (2006)
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