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

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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 ...

Our protocol can be useful in the systems and mechanism. And the right information shows the effect of physical stress and massage. Our local cyclic completion system is much more simple.The conventional methods aren't required for local insertion. Apply surgical tape to the bilateral hind limbs in the supine position with the knee joints extended and the ankle joints plantar flexed. Place an aluminum wire on the trunk at the L4-5 spine level, and coil the wire in a spiral configuration around the hind limbs with five millimeter gaps between each turn of the spiral.To minimize the possibility of escape from the wiring, manually adjust the wire configuration to immobilize the hip joints at a 90 degree position of abduction. Then return the mouse to it's home cage with monitoring until full recovery. To measure the intramuscular gastrocnemius muscle pressure place the anesthetized mouse in the prone position and shave, depilate, and semi-sterilize the skin of the posterior calf.Use a scalpel to make a two millimeter incision in the disinfected skin. And insert a 20 gauge indwelling needle into the gastrocnemius muscle at a 150 to 170 degree angle to the skin surface. Next, using the plastic sheath of the needle as a guide, place a blood pressure telemeter sensor in the mid belly of gastrocnemius muscle.Enclose the skin with a 4-O nylon suture. Then, use several different cylindrical unit weights to apply local cylindrical compression to the calf muscle for one minute. And monitor the intramuscular pressure using the appropriate software for biological signal analysis.For local cyclical compression treatment disengage the anesthetized mouse from the hind limb wiring. And place the animal in the prone position. Adjust the hind limbs so that the knee joints are extended and the ankle joints are plantar flexed with the calves facing upward.And vertically move a cylindrical weight unit, covered with a cushioned pad, over the calf muscle at one hertz for 30 minutes per day for seven days. After each daily bout of compression rewire the mouse hind limbs before returning the animal to its home cage. At the end of the experiment depilate the posterior calf surface and make a skin incision to allow separation of the gastrocnemius muscles from the tibiofibular bone.Quickly freeze the harvested muscle tissue in an optimal cutting temperature compound solution. And use a cryostat to obtain sections of the gastrocnemius muscles on glass slides. For immunohistochemical labeling thaw and dehydrate the sections by air drying at room temperature before using a hydrophobic pen to draw a ring around the tissue sections on each slide.Next, place the slides in humidity chamber. And add 100 microliters of blocking buffer to each side. After 30 minutes at room temperature rinse the slides with two 5 minute washes in PBS-T, before labeling the sections with 100 microliters of the primary antibody of interest overnight at room temperature.The next morning wash the slides 3 times in PBS-T for 5 minutes per wash. And label the samples with an appropriate secondary antibody for 1 hour at room temperature, protected from light. At the end of the incubation, wash the slides 3 times in fresh PBS-T as demonstrated.And stain the nuclei with 100 microliters of DAPI for three minutes at room temperature in the dark. Then, wash the slide three more times. And mount the samples with a compatible mounting medium and cover slips.For histomorphometric analysis, place the slides on the stage of a fluorescence microscope and view the samples under a 20-X subjective in the appropriate filters. Then, in the image analysis software measure the cross sectional area of each myofiber and count the number of cells expressing the experimental markers of interest. The cross sectional area of gastrocnemius myofiber are significantly decreased by hind limb immobilization.Further, immunofluorescent staining analysis reveals that cells expressing MCP-1 and TNF-alpha are significantly increased in the gastrocnemius muscle tissues of immobilized hind limbs. Together, with the increase in cells positively stained with F4/80, hind limb immobilization appears to instigate calf muscle atrophy, involving local inflammatory responses, including macrophage accumulation. Among the several different local cyclical compression magnitudes tested by changing the weight of the cylindrical unit, the compression corresponding to 50 milliliters of mercury intermuscular pressure waves, appears to most efficiently alleviate the immobilization induced decrease in myofiber cross sectional area and to increase in the macrophage accumulation in gastrocnemius muscles.A 66 gram local cyclical compression, applied at 1 hertz for 30 minutes per day for 7 days, significantly alleviates the observed immobilization induced decreases in the myofiber cross sectional area of the gastrocnemius muscles. More, local cyclical compression partially tempers immobilization induced decreases in the contracting force of the triceps surae muscles. In addition, local cyclical compression tempers the increase in F4/80 positive, TNF-alpha positive, and MCP-1 one positive cells in the gastrocnemius muscle tissue of immobilized hind limbs.Our approach will provide a scientific basis for muscle rise in the patient and our potential strategy for preventive procedure. The rat's found the limited of physical inactivity. Using our system, we have started assessing the most patients that's muscles lack mechanic in the patient.Moderate local industrial way to do it makes a perfect immobilization induced muscle atrophy.

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