Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

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 alike^1,2. In fact, previous studies have shown that massage suppresses local inflammation³ and prompts recovery from the post-exercise muscle damage^4,5. 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 ....

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 processes^13,14, 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,.......

Materials

Name	Company	Catalog Number	Comments
Aluminum wire	DAISO JAPAN	B028	An aluminum wire is used to avoid escaping restriction by the wire
Blood pressure telemeter	Millar	SPR-671	A blood pressure telemeter is used to mesure intramuscular pressure.
DAPI	Thermo Fisher Scientific	D1306	DAPI 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	A11034	Antibody for immunohistochemical staining.
Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500))	Invitrogen	A11077	Antibody for immunohistochemical staining.
ImageJ	NIH	N/A	Analysis software for image
LabChart8	ADInstrumens		Analysis software for acquiring biological signals.
Prolong gold	Thermo Fisher Scientific	P36930	Prolong gold is for mounting stained samples.
Protein Block Serum-Free	Dako	X090930-2	For blocking non-specific background staining in immunohistochemical procedures.
Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000)	Sigma Aldrich	L0663	Antibody for immunohistochemical staining.
Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500)	Abcam	ab6640	Antibody for immunohistochemical staining.
Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000)	Abcam	ab25124	Antibody for immunohistochemical staining.
Rabbit polyclonal anti-TNF-α antibody (Dilution ratio, 1:1000)	Abcam	ab66579	Antibody for immunohistochemical staining.
Surgical tape	3M Japan	1530EP-0	Surgical tape is used to restrict joint movement.