Name: application of consistent massage-like perturbations on mouse calves and monitoring the resulting intramuscular pressure changes
Uploaded: 2019-09-20T13:00:00
Description: Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

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).

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.
<ol>
	<li>Adequately anesthetize a mouse using sodium pentobarbital (50 mg/kg i.p.). Make sure that mice do not respond to ...

<ol>
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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,...

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 experimental techniques by which massage-like interventions are tested. In previous studies, experimental procedures that mimic massage mostly involve the application of physical interventions using practitioners&rsquo; body parts, such as palms and fingers6,7,8. This makes it is difficult to precisely reproduce their magnitude, frequency, duration, and mode.
Many devices have been developed to apply defined mechanical loads to the target tissues. For example, Zeng et al. have developed a pneumatic system for the length-wise mechanical loading to rats&rsquo; hindlimbs9 and Wang et al. have developed a mechatronic device that can apply massage-like mechanical loads to hindlimbs of rats and rabbits with real-time feedback control10. Compared to them, our local cyclical compression (LCC) system is much simpler, demanding far less cost for construction. Nonetheless, we can reproduce the intramuscular pressure changes that are generated during the mild muscle contraction. Using this device, we have successfully demonstrated that the massage-like mechanical interventions modulate local interstitial fluid dynamics and alleviate immobilization-induced muscle atrophy11.
Here, we describe the details of our device and the protocol, which may help explore the molecular mechanisms behind the positive effects of exercises and massages. The schematics of the protocol is presented as Supplementary Figure 1.

<table>
<tbody>
<tr>
<td>Aluminum wire</td>
<td>DAISO JAPAN</td>
<td>B028</td>
<td>An aluminum wire is used to avoid escaping restriction by the wire</td>
</tr>
<tr>
<td>Blood pressure telemeter</td>
<td>Millar</td>
<td>SPR-671</td>
<td>A blood pressure telemeter is used to mesure intramuscular pressure.</td>
</tr>
<tr>
<td>DAPI</td>
<td>Thermo Fisher Scientific</td>
<td>D1306</td>
<td>DAPI is a fluorescent probe which is commonly used to stain DNA for fluorescent microscopy.</td>
</tr>
<tr>
<td>Goat anti-rabbit Alexa Fluor 488 (Dilution ratio, 1:500)</td>
<td>Invitrogen </td>
<td>A11034</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500))</td>
<td>Invitrogen </td>
<td>A11077</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>ImageJ</td>
<td>NIH</td>
<td>N/A</td>
<td>Analysis software for image</td>
</tr>
<tr>
<td>LabChart8</td>
<td>ADInstrumens</td>
<td>&nbsp;</td>
<td>Analysis software for acquiring biological signals.</td>
</tr>
<tr>
<td>Prolong gold</td>
<td>Thermo Fisher Scientific</td>
<td>P36930</td>
<td>Prolong gold is for mounting stained samples.</td>
</tr>
<tr>
<td>Protein Block Serum-Free</td>
<td>Dako</td>
<td>X090930-2</td>
<td>For blocking non-specific background staining in immunohistochemical procedures.</td>
</tr>
<tr>
<td>Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000)</td>
<td>Sigma Aldrich</td>
<td>L0663</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500)</td>
<td>Abcam</td>
<td>ab6640</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab25124</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-TNF-&alpha; antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab66579</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Surgical tape</td>
<td>3M Japan</td>
<td>1530EP-0</td>
<td>Surgical tape is used to restrict joint movement.</td>
</tr>
</tbody>
</table>

application of consistent massage-like perturbations on mouse calves and monitoring the resulting intramuscular pressure changes

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.

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-&#945;, both of which play key roles in regulating inflammatory processes13,14, significantly increased in gastrocnemius muscle tissues of immobil...

Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

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

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.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

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