A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Abstract

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Introduction

Massage is generally recognized to be beneficial for both pain relief and improvement of the physical performance among competitive athletes and non-athletes alike1,2. In fact, previous studies have shown that massage suppresses local inflammation3 and prompts recovery from the post-exercise muscle damage4,5. Molecular mechanisms underlying the beneficial effects of massage remain largely unknown.

One of the difficulties with the mechanistic investigation on massage relates to the reproducibility of e....

Protocol

All animal experiments were conducted under the approval by the Institutional Animal Care and Use Committee of the National Rehabilitation Center for Persons with Disabilities.

1. Immobilization of the mouse bilateral hindlimbs

NOTE: Male C57BL/6 mice were used for experiments at the age of 11 - 12 weeks after acclimation for at least 7 days.

  1. Adequately anesthetize a mouse using sodium pentobarbital (50 mg/kg i.p.). Make sure that mice do not respond to a hindlimb toe pinch.
    NOTE: Conduct the procedure of immobilization between 10 a.m. and 7 p.m. to minimize the possible effects on the feeding ....

Representative Results

Consistent with our previous observations12, the CSA of gastrocnemius myofibers were significantly decreased by hindlimb immobilization (Figure 2A,B). Furthermore, our immunofluorescence staining analysis revealed that cells expressing MCP-1 and TNF-α, both of which play key roles in regulating inflammatory processes13,14, significantly increased in gastrocnemius muscle tissues of immobil.......

Discussion

We have described a method for applying a massage-like mechanical stimulus, which has anti-inflammatory effects. Our system has following advantages even when compared with those reported previously. First, previous studies did not quantitatively define the mechanical forces applied2 or defined their magnitudes based on the measurement at the body surface, but not inside the tissues10. In contrast, we measured intramuscular pressure using a blood pressure telemeter. Second,.......

Disclosures

The authors declare that there are no competing interests associated with the manuscript.

Acknowledgements

We thank K. Nakanishi, K. Hamamoto, N. Kume, and K. Tsurumi for their consistent support throughout the project. This work was in part supported by Intramural Research Fund from the Japanese Ministry of Health, Labour and Welfare; Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015-2019 from the Japanese Ministry of Education, Culture, Sports, Science and Technology (S1511017).

....

Materials

NameCompanyCatalog NumberComments
Aluminum wireDAISO JAPANB028An aluminum wire is used to avoid escaping restriction by the wire
Blood pressure telemeterMillarSPR-671A blood pressure telemeter is used to mesure intramuscular pressure.
DAPIThermo Fisher ScientificD1306DAPI is a fluorescent probe which is commonly used to stain DNA for fluorescent microscopy.
Goat anti-rabbit Alexa Fluor 488 (Dilution ratio, 1:500)Invitrogen A11034Antibody for immunohistochemical staining.
Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500))Invitrogen A11077Antibody for immunohistochemical staining.
ImageJNIHN/AAnalysis software for image
LabChart8ADInstrumens Analysis software for acquiring biological signals.
Prolong goldThermo Fisher ScientificP36930Prolong gold is for mounting stained samples.
Protein Block Serum-FreeDakoX090930-2For blocking non-specific background staining in immunohistochemical procedures.
Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000)Sigma AldrichL0663Antibody for immunohistochemical staining.
Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500)Abcamab6640Antibody for immunohistochemical staining.
Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000)Abcamab25124Antibody for immunohistochemical staining.
Rabbit polyclonal anti-TNF-α antibody (Dilution ratio, 1:1000)Abcamab66579Antibody for immunohistochemical staining.
Surgical tape3M Japan1530EP-0Surgical tape is used to restrict joint movement.

References

  1. Furlan, A. D., Imamura, M., Dryden, T., Irvin, E. Massage for low back pain: an updated systematic review within the framework of the Cochrane Back Review Group. Spine. 34 (16), 1669-1684 (2009).
  2. Robertson, A., Watt, J. M., Galloway, S. D. R.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

MassagePerturbationsIntramuscular PressureMouse CalfPhysical StressLocal Cyclic CompressionGastrocnemius MusclePressure MonitoringAnesthetized MouseHind Limb WiringMuscle Tissue Harvesting

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved