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 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 activity of mice.</li>
	<li>Apply surgical tapes to the bilateral hindlimbs of the mouse laid in a supine position with the knee joints extended and ankle joints plantar-flexed.</li>
	<li>Place an aluminum wire (see Table of Materials) on the trunk at L4-5 spine level and coil the wire in a spiral configuration around the hindlimbs with 5 mm gaps between each turn of the spiral layer (Figure 1A). Make sure not to coil the wire too tightly and avoid disturbing the local blood flow.</li>
	<li>To minimize the possibility of escape from wiring, immobilize the hip joints at the position of 90&#176; abduction by manually adjusting the configuration of aluminum wire.</li>
	<li>Return the mice to their original cages. 3 h later, make sure that they recover from anesthesia and access to food and water as usual.</li>
	<li>House 3 - 6 immobilized mice per cage as before immobilization.</li>
</ol>
2. Measurement of intramuscular pressure of mouse gastrocnemius muscles
NOTE: Several different weights of cylindrical units (36 g, 66 g, and 200 g) were tested in the pressure-monitoring experiments combined with LCC. This measurement was conducted separately from the experiments to analyze muscle inflammation and atrophy (see step 3 - 5 for more details) i.e., the mice subjected to pressure measurement were not used for histological analyses.
<ol>
	<li>Because pressure measurement involves more invasive procedures (e.g., skin incision and needle insertion) as compared to hindlimb wiring and LCC, use a mixture of three anesthetic agents (medetomidine 0.75 mg/kg, midazolam 4.0 mg/kg, and butorphanol 5.0 mg/kg, i.p.). Make sure that mice do not respond to the hindlimb toe pinch.</li>
	<li>Lay the mouse in a prone position, make a 2-mm incision with a scalpel on the posterior calf after depilating with an electric shaver and semi-sterilizing the skin surface with 70% ethanol- and Chlorhexidine-soaked absorbent cotton.</li>
	<li>Insert a 20 G indwelling needle into the gastrocnemius muscle at an obtuse angle (150&#176; &#8211; 170&#176;) to the skin surface.</li>
	<li>Using the plastic sheath of the needle as a guide, place a sensor of the blood pressure telemeter (see Table of Materials) in the mid-belly of gastrocnemius muscle, and then remove the sheath from the muscle.</li>
	<li>After suturing the skin with 4-0 nylon suture, apply LCC with several different weights of cylindrical units to the calf in the mice (see step 3 for more details), and monitor the intramuscular pressure using software for biological signal analysis (see Table of Materials).</li>
	<li>Return the mice to their original cages. 3 h later, make sure that they recover from anesthesia/analgesia&#160;and have access to food and water as usual.</li>
</ol>
3. Local cyclical compression (LCC) on mouse calves
<ol>
	<li>Except for the intramuscular pressure measurement and euthanizing (i.e., cervical dislocation), use sodium pentobarbital (50 mg/kg i.p.) for anesthesia.</li>
	<li>Disengage the mouse from hindlimb wiring and lay it in a prone position with the knee joints extended and the ankle joints plantar-flexed so that the calves faced upward. Do not fix the mouse hindlimbs on the stage.</li>
	<li>Apply LCC to the calf by vertically moving a cylindrical weight unit (Figure 1B) covered with a cushion pad (Figure 1C) at 1 Hz for 30 min per day, 7 days.</li>
	<li>After each bout of daily LCC, re-wire the mouse hindlimbs.</li>
</ol>
4. Immunohistochemical analysis of gastrocnemius
<ol>
	<li>Euthanize the mouse by cervical dislocation under adequate anesthesia/analgesia by intraperitoneal injection of a mixture of three anesthetic agents (medetomidine 0.75 mg/kg, midazolam 4.0 mg/kg, and butorphanol 5.0 mg/kg).</li>
	<li>After depilating the posterior calf surface, make a skin incision, and dissect gastrocnemius muscles by separating from tibio-fibular bone using a surgical scissor and quickly freeze them in an optimal cutting temperature compound solution.</li>
	<li>Using a cryostat, prepare cryo-section samples of gastrocnemius muscles on glass slides. Store the samples in a -80 &#176;C freezer until analysis.</li>
	<li>Take out the gastrocnemius cryo-section samples to be analyzed from the freezer and dehydrate them by air drying at room temperature.</li>
	<li>Use a liquid blocker pen to draw an area that includes all the cryo-sections on the slide. The circle will prevent the solutions from flowing off the slide.</li>
	<li>Avoid drying of the samples by placing the slides in a tray in which a moist environment is created with water-soaked paper cloth.</li>
	<li>Apply 100 &#181;L of blocking buffer (phosphate-buffered saline (PBS) containing 0.25% casein, carrier protein, and 0.015 M sodium azide) for 30 min at room temperature.</li>
	<li>Rinse the slides twice by incubating with PBS-T (PBS containing 0.1% polyoxyethylene sorbitan monolaurate (see Table of Materials) for 5 min.</li>
	<li>Apply 100 &#181;L of primary antibody diluted with PBS on each sample, cover the tray with a lid, and incubate overnight at room temperature.</li>
	<li>Wash 3 times with PBS-T (5 min for each wash).</li>
	<li>Apply 100 &#181;L of secondary antibody diluted with PBS on each sample and incubate for 1 h at room temperature. 
	NOTE: For anti-laminin staining, use Alexa Fluor 568-conjugated secondary antibody. For anti-F4/80, anti-MCP-1, and anti-TNF-&#945;, use Alexa Fluor 568- or 488-conjugated secondary antibody.</li>
	<li>Wash 3 times with PBS-T (5 min for each wash).</li>
	<li>Apply 100 &#181;L of DAPI solution diluted with PBS-T on each sample and incubate for 3 min at room temperature.</li>
	<li>Wash 3 times with PBS-T (5 min for each).</li>
	<li>Mount the samples with mounting medium and cover them with coverslips.</li>
</ol>
5. Histo-morphometric analysis of gastrocnemius
<ol>
	<li>Place the sample slides on a fluorescence microscope (see Table of Materials) and view the samples using a 20&#215; objective with appropriate filters (DAPI-B, 360/40 nm for excitation and 460/50 nm for emission; GFP-B, 470/40 nm for excitation and 535/50 nm for emission; FRITC, 540/25 nm for excitation and 605/55 nm for emission.</li>
	<li>Using the software for image analysis (see Table of Materials), measure the cross-sectional area (CSA) of each myofiber, and count the number of F4/80-, MCP-1-, and TNF-&#945;-positive cells. 
	NOTE: Determine CSA of each myofiber by tracing the internal margin of the basement membrane visualized with anti-laminin-2 immunostaining.</li>
</ol>

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	<li>Furlan, A. D., Imamura, M., Dryden, T., Irvin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+for+low+back+pain:+an+updated+systematic+review+within+the+framework+of+the+Cochrane+Back+Review+Group.">Massage for low back pain: an updated systematic review within the framework of the Cochrane Back Review Group.</a> Spine. 34 (16), 1669-1684 (2009).</li><li>Robertson, A., Watt, J. M., Galloway, S. D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+leg+massage+on+recovery+from+high+intensity+cycling+exercise.">Effects of leg massage on recovery from high intensity cycling exercise.</a> British Journal of Sports Medicine. 38 (2), 173-176 (2004).</li><li>Waters-Banker, C., Butterfield, T. A., Dupont-Versteegden, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+massage+on+nonperturbed+skeletal+muscle+in+rats.">Immunomodulatory effects of massage on nonperturbed skeletal muscle in rats.</a> Journal of Applied Physiology. 116 (2), 164-175 (2014).</li><li>Haas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+timing+affects+postexercise+muscle+recovery+and+inflammation+in+a+rabbit+model.">Massage timing affects postexercise muscle recovery and inflammation in a rabbit model.</a> Medicine &amp; Science in Sports &amp; Exercise. 45 (6), 1105-1112 (2013).</li><li>Crane, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+therapy+attenuates+inflammatory+signaling+after+exercise-induced+muscle+damage.">Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage.</a> Science Translational Medicine. 4 (119), 119ra113 (2012).</li><li>Bove, G. M., Harris, M. Y., Zhao, H., Barbe, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+therapy+as+an+effective+treatment+for+fibrosis+in+a+rat+model+of+upper+extremity+overuse+injury.">Manual therapy as an effective treatment for fibrosis in a rat model of upper extremity overuse injury.</a> Journal of the Neurological Sciences. 361, 168-180 (2016).</li><li>Andrzejewski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+skeletal+muscle+expression+of+VEGF+induced+by+massage+and+exercise.">Increased skeletal muscle expression of VEGF induced by massage and exercise.</a> Folia Histochemica et Cytobiologica. 53 (2), 145-151 (2015).</li><li>Mantovani Junior, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+massage+as+a+recuperative+technique+on+autonomic+modulation+of+heart+rate+and+cardiorespiratory+parameters:+a+study+protocol+for+a+randomized+clinical+trial.">Effects of massage as a recuperative technique on autonomic modulation of heart rate and cardiorespiratory parameters: a study protocol for a randomized clinical trial.</a> Trials. 19 (1), 459 (2018).</li><li>Zeng, H., Butterfield, S., Agarwal, F., Haq, T., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineering+approach+for+quantitative+analysis+of+the+lengthwise+strokes+in+massage+therapies.">An engineering approach for quantitative analysis of the lengthwise strokes in massage therapies.</a> Journal of Medical Devices. 2 (4), (2008).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechatronic+system+for+quantitative+application+and+assessment+of+massage-like+actions+in+small+animals.">A mechatronic system for quantitative application and assessment of massage-like actions in small animals.</a> Annals of Biomedical Engineering. 42 (1), 36-49 (2014).</li><li>Saitou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+cyclical+compression+modulates+macrophage+function+in+situ+and+alleviates+immobilization-induced+muscle+atrophy.">Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy.</a> Clinical Science. 132 (19), 2147-2161 (2018).</li><li>Onda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+mouse+model+of+skeletal+muscle+atrophy+using+spiral+wire+immobilization.">A New mouse model of skeletal muscle atrophy using spiral wire immobilization.</a> Muscle Nerve. 54 (4), 788-791 (2016).</li><li>Luster, A. 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The authors declare that there are no competing interests associated with the manuscript.

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>&amp;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-&amp;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 ...

application-consistent-massage-like-perturbations-on-mouse-calves

Research

JoVE Journal

Immunology and Infection

5.6K Views.  National Rehabilitation Center for Persons with Disabilities. Ons protocol kan nuttig zijn in de systemen en het mechanisme. En de juiste informatie toont het effect van fysieke stress en massage. Ons lokale cyclische voltooiingssysteem is veel eenvoudiger.De conventionele methoden zijn niet vereist voor lokale invoeging. Breng chirurgische tape aan op de bilaterale achterpoten in de supinepositie met de verlengde kniegewrichten en de enkelgewrichten plantar gebogen. Plaats een aluminium draad op de romp op de L4-5 wervelkolom niveau, en spoel de...

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

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

Behavior

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Neuroscience

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Medicine

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Biology

Fixation and Operational Method for Abdominal Massage in T2DM Mice

Type 2 diabetes mellitus (T2DM) is a rapidly growing global public health issue, affecting over 500 million people worldwide. Although abdominal massage has shown potential benefits in managing T2DM, its effectiveness remains unclear, particularly in animal studies, where challenges such as animal compliance and the need for precise pressure control complicate implementation. To address these challenges, this study introduces a novel abdominal massage simulation device specifically designed for mice. This device provides a practical solution for conducting abdominal massage interventions in a controlled manner while minimizing stress and tissue damage to the animals. It securely restrains the mice's limbs, allowing them to remain conscious during the massage, and offers precise control over both the pressure and frequency of the massage applied to the abdomen. The device's ability to simulate manual abdominal massage with accuracy opens new possibilities for experimental studies assessing its effects on T2DM. The primary goal of this protocol is to investigate the impact of abdominal massage on key T2DM markers such as blood glucose levels, lipid metabolism, and insulin sensitivity in mice. By providing a reliable and reproducible method for abdominal massage, this device can offer valuable insights into its potential as a non-invasive therapeutic intervention for T2DM. The findings from this research may contribute to advancing clinical strategies for diabetes prevention and treatment, particularly in enhancing the understanding of traditional therapies like abdominal massage in modern medical practice.

This study introduces an abdominal massage device for mice that replicates manual massage while minimizing stress and tissue damage. The device significantly lowers blood glucose levels, improves lipid metabolism, and enhances insulin sensitivity in type 2 diabetes mice, offering a promising, non-invasive therapeutic approach with clinical potential.

Type 2 diabetes mellitus (T2DM) is a rapidly growing global public health issue, affecting over 500 million people worldwide. Although abdominal massage ...

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

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