Name: dosage-adjusted resistance training in mice with a reduced risk of muscle damage
Uploaded: 2022-08-31T14:20:00
Description: Watch this Scientific Journal Video about Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage at JoVE.com

This study was funded by grants from the Jain Foundation Inc., R03HD091648 from NICHD, a Pilot Grant from AR3T under NIH P2CHD086843, a FRAP Award from EACPHS at Wayne State University, a Faculty Startup Package from Wayne State University, and a subcontract from 1R01AR079884-01 (Peter L. Jones PI) to JAR. This study was also funded by an American Physical Therapy Association - Michigan (APTA-MI) research grant to JMB, MEP, and JAR. The authors acknowledge Dr. Renuka Roche (Associate Professor, Eastern Michigan University, MI) for critically reading the manuscript and providing feedback. The authors acknowledge Mr. Anselm D. Motha for advice on 3D printing. The authors thank the patients with dysferlinopathies who have shared their stories on the Jain Foundation website at https://www.jain-foundation.org/patient-physician-resources/patient-stories,&#160;particularly their experiences with exercise.

The experiments described in this article were approved by the Institutional Animal Care and Use Committee (IACUC) at Wayne State University, Detroit, Michigan, USA, in accordance with the Guide for the Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA). B6.A-Dysfprmd/GeneJ mice (a.k.a. BLAJ mice, males, ~1.5 years old) that model LGMD2B/R2 were used for the present study. The mice were obtained from a commercial source ...

This article presents step-by-step instructions on how to construct a device to perform a type of precision rehabilitation training called dosage-adjusted resistance training (DART). The work also describes the application of the DART device and methodology in a training study to compare muscle damage 3 days after a single bout of DART (DART group) with damage 3 days after a comparable bout of isometric training (ISOM group).
The critical steps in the protocol are the proper construction of th...

Exercise confers numerous health benefits on skeletal muscle (reviewed in Vina et al.1). Specifically, progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads (e.g., barbells, dumbbells, cable-pulley-weight circuits), is known to help increase muscle mass and strength in both healthy individuals and patient populations (reviewed in previous publications2,3). PRT is based on the overload principle, which states that, when the muscle contracts against progressively greater external loads, it adapts by increasing its physiological cross-sectional area as well as force-producing capacity4. Existing models of PRT in rodents include ladder climbing with resistance applied to the tail, co-contraction of agonist muscles against resistance from antagonists, running with a weighted harness, a squatting exercise elicited by an electrical shock, and resisted wheel-running5,6,7,8,9,10 (reviewed in previous publications11,12). However, there are currently no research tools to perform precisely muscle-targeted, dosage-adjusted PRT in mice that closely resemble the PRT methods and devices used in human clinical research and practice12,13. This limits the ability of investigators to study the safety and effectiveness of precisely dosed PRT in basic and preclinical studies in mice.
To overcome this barrier, a PRT methodology and device are developed in this study based on the cable-pulley-weight circuit designs employed in resistance training equipment in modern gymnasiums14,15,16. This method of PRT is referred to as dosage-adjusted resistance training (DART), and the device is called the DART device. In addition to its functionality as a precision rehabilitation training tool, the DART device can also be used as a standalone instrument to objectively assess the maximum concentric contractile torque that can be generated by the tibialis anterior (TA) muscle in a mouse, similar to how the one-repetition maximum (1RM, the maximum load that can be successfully lifted/moved/pressed/squatted just one time while maintaining good form) is assessed in humans17,18. The DART device can also be coupled with a custom-built or commercial isokinetic dynamometer to measure the peak isometric tetanic force produced by the TA muscle in a mouse (comparable to maximum voluntary contraction [MVC] in humans) and then perform dosage-adjusted PRT with a resistance that is based on the peak tetanic force (e.g., 50% of the peak force).
This article describes the construction of the DART device and explains how it can be coupled with a custom-built dynamometer, which has been described in prior publications19,20,21,22, to assess contractile torque and perform DART. The study also describes how the DART device was used to compare exercise-induced muscle damage caused by a single bout of DART (4 sets of 10 concentrically biased contractions with 50% 1RM) to damage caused by a comparable bout of isometric contractions (4 sets of 10 isometric contractions) in a mouse model of limb-girdle muscular dystrophy type 2B (LGMD2B, or LGMDR2)23,24. The mouse model that was studied lacks a protein called dysferlin, which plays an important role in protecting skeletal muscle against delayed-onset muscle damage following injurious eccentric contractions22,25,26,27,28,29,30. It has also been demonstrated in dysferlin-deficient male mice that concentrically biased forced exercise is not as damaging as eccentrically biased forced exercise and that prior exposure to concentrically biased training offers protection against injury from a subsequent bout of eccentrically biased contractions22. Since the current study was conducted to test the feasibility of the present DART methodology and device in performing dosage-adjusted, concentrically biased resistance training, male dysferlin-deficient mice were chosen for the investigation to compare new data from the DART device with previous data. In future studies, female BLAJ mice will be included to study the effect of sex as a biological variable in relation to the response to DART. Mice that were ~1.5 years old were studied since they already have dystrophic changes in many muscle groups and, therefore, model the pathophysiological state in which muscles might be in patients who already have muscle weakness and wasting and are seeking rehabilitative care to maintain muscle mass and strength26.

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			<td>AnMiao Star 608 Ceramic Ball Bearing</td>
			<td>Anmiao Star (N/A)</td>
			<td>AMS127</td>
			<td>High precision, low friction wheel bearing.&#160; If make and model is not commercially available, an alternative version of a 608 low-friction wheel bearing, 8 mm bore diameter,&#160; 22 mm outside diameter, with silicon nitride ceramic balls in 420 stainless steel housing should suffice.&#160; Excess friction in the wheel bearing will adversely impact performance of the DART device and will increase overall resistance to muscle contractions.</td>
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		<tr>
			<td>Axio Scope.A1 microscope</td>
			<td>Carl Zeiss (Peabody, MA)</td>
			<td>Product #Axio Scope.A1</td>
			<td>Light and fluorescence microscope</td>
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		<tr>
			<td>B6.A-Dysfprmd/GeneJ (a.k.a. BLAJ mice)</td>
			<td>The Jackson Laboratory (Bar Harbor, ME).&#160; Special colony maintained by The Jain Foundation Inc. for collaborators who study dysferlin.</td>
			<td>Stock #012767</td>
			<td>Dysferlin deficient mice that model human limb girdle muscular dystrophy type 2B/R2.</td>
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			<td>Bipolar, transcutaneous, neuromuscular electrical stimulation (NMES) electrode</td>
			<td>Harvard Apparatus, Holliston, MA</td>
			<td>BS4 50&#8211;6824</td>
			<td>Electrode for NMES.&#160; If this electrode is not commercially available, please contact corresponding author for alternatives.</td>
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			<td>Coplin Staining Dish</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog No. S17495</td>
			<td>Staining dish/jar for hematoxylin and eosin (H&#38;E) staining of sections</td>
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			<td>Cura 4.4.1. Software</td>
			<td>Ultimaker, Utrecht, Netherlands</td>
			<td>Ultimaker Cura 4.4.1.</td>
			<td>Slicing software to convert stereolithography files into G-CODE files</td>
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			<td>Deltaphase isothermal gel heating pad</td>
			<td>Braintree Scientific (Braintree, MA)</td>
			<td>Item #39DP</td>
			<td>Heating pad to provide thermal support to animals while under anesthesia</td>
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			<td>Eosin Y</td>
			<td>Millipore Sigma (Burlington, MA)</td>
			<td>HT110132-1L</td>
			<td>Pink cytoplasmic stain</td>
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			<td>Gorilla Super Glue</td>
			<td>The Gorilla Glue Company (Cincinnati, OH)</td>
			<td>Gorilla Super Glue Micro Precise</td>
			<td>Cyanoacrylate adhesive to bond PLA components</td>
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			<td>Hematoxylin solution, Gill No.3</td>
			<td>Millipore Sigma (Burlington, MA)</td>
			<td>GHS332-1L</td>
			<td>Dark blue stain for nuclei</td>
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			<td>HM525NX cryostat</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog #HM525NX</td>
			<td>Cryostat to make frozen sections of muscle</td>
		</tr>
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			<td>Lab Wipes.&#160; Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>Catalog No. 06-666.&#160; Manufacturer #34120</td>
			<td>Laboratory wipes to blot mineral oil from muscle tissue before snap freezing and for other purposes.</td>
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			<td>Labview 2014</td>
			<td>National Instruments, Austin, Texas, USA</td>
			<td>Labview 2014</td>
			<td>Software for custom-written programs/routines that operate the dynamometer and trigger the NMES stimulator.</td>
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			<td>Liquid nitrogen HDPE Dewar Flasks</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>S34074B.&#160; Thermo Scientific 41502000/EMD</td>
			<td>Flask to hold liquid nitrogen for snap freezing muscle or other tissue</td>
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			<td>Magic depilatory cream</td>
			<td>Softsheen Carson (New York, NY)</td>
			<td>N/A</td>
			<td>Razorless hair removal cream</td>
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			<td>Metal alligator clip</td>
			<td>JINSHANGTOPK (web-based business)</td>
			<td>24Pcs 51mm Metal Alligator Clip Spring Clamps</td>
			<td>Spring clamp to hold tibial pin</td>
		</tr>
		<tr>
			<td>Micrscope slides</td>
			<td>Globe Scientific (Mahwah, NJ)</td>
			<td>1354W. Diamond White Glass Slides</td>
			<td>Charged microscope slides</td>
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			<td>Mineral Oil</td>
			<td>ThermoFisher (Waltham, MA)</td>
			<td>BP26291</td>
			<td>Mineral oil to cryoprotect muscle tissue before snap freezing</td>
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			<td>Monoprice Premium 3D Printer Filament PLA</td>
			<td>Monoprice (Rancho Cucamonga, CA)</td>
			<td>#11778</td>
			<td>Premium 3D Printer Filament PLA 1.75mm 1 kg/spool, Gray.&#160; This is the material used to 3D print device components.</td>
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			<td>Monoprice Select Mini V2 3D printer</td>
			<td>Monoprice (Rancho Cucamonga, CA)</td>
			<td>Mini V2 3D</td>
			<td>3D printer for computer-aided fabrication of device components.</td>
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			<td>NIH Image software</td>
			<td>National Instritues of Health (NIH, Bethesda, MD)</td>
			<td>NIH Image for Windows</td>
			<td>Image processing and analysis software used to quantify area of muscle damage.&#160; NIH Image is also known as Image J.</td>
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			<td>Photoshop CS4</td>
			<td>Adobe (San Jose, CA)</td>
			<td>Creative Suite (CS4). 64 bit version for Windows</td>
			<td>Image processing and analysis software used to generate tiled/stiched images of entire muscle cross-section from images of indvidual overlapping fields</td>
		</tr>
		<tr>
			<td>PSIU6 stimulation isolation unit</td>
			<td>Grass Instruments (West Warwick, RI)</td>
			<td>PSIU6 isolation unit</td>
			<td>Isolation unit for NMES.&#160; Stimulators, such as Model 4100 from A-M come with a built in stimulation isoloation unit</td>
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			<td>Roboz 4-0 silk black braided suture material</td>
			<td>Roboz Surgical (Gaithersburg, MD)</td>
			<td>Roboz Surgical SUT152</td>
			<td>Suture material to connect DART device footplate to dynamometer footplate or resistance for resistance training</td>
		</tr>
		<tr>
			<td>S48 square pulse stimulator</td>
			<td>Grass Instruments (West Warwick, RI)</td>
			<td>S48 Stimulator</td>
			<td>Laboratory electrical stimulator for NMES .&#160; If this stimulator is not commercially available, Model 4100 Isolated High Power Stimulator from A-M systems could be an alternative.&#160; Please contact co-author Jones for more information.</td>
		</tr>
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			<td>Scott&#8217;s bluing reagent</td>
			<td>Ricca Chemical Company (Arlington, TX)</td>
			<td>6697-32</td>
			<td>Bluing solution that intensifies hematoxylin nuclear staining</td>
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			<td>SigmaStat version 3.5</td>
			<td>Systat Software (San Jose, CA)</td>
			<td>SigmaStat version 3.5</td>
			<td>Statistical software package for statistical analyses</td>
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			<td>Tabletop isoflurane vaporizer</td>
			<td>VetEquip (Livermore, CA)</td>
			<td>Item #901801</td>
			<td>Inhaled tabletop anesthesia system</td>
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			<td>Triple antibiotic first aid ointment</td>
			<td>Global Health Products (wed-based business)</td>
			<td>Globe Triple Antibiotic First Aid Ointment, 1 oz (2-Pack) First Aid Antibiotic Ointment</td>
			<td>Antibiotic ointment applied on tibial pin as part of post-procedural care</td>
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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.

BLAJ male mice, which were ~1.5 years in age, were studied. BLAJ mice model the human muscle disease, LGMD2B/R2. These mice are particularly susceptible to delayed onset muscle damage from a single bout of eccentric muscle contractions22,29. BLAJ mice were, therefore, chosen for these studies to learn if DART could be performed in a non-injurious manner by precisely adjusting the resistance against which the TA muscle has to work in a concentrically biased manner...

Watch this Scientific Journal Video about Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage at JoVE.com

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

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 ...

This protocol is one of the first studies that enables scientists to perform dosage-adjusted resistance training in mice similar to how it's performed in humans. The main advantage of this technique is that it allows investigators to precisely adjust resistance against which the mouse's anterior tibial muscles have to work during resistance training. Furthermore, since muscle contractions are eccentrically biased, there is a lower chance of contraction-induced muscle injury.Our dosage-adjusted resistance training technique can be incorporated into basic and preclinical studies in mice to develop interventions for the improvement or maintenance of muscle mass and strength across a diverse array of disease conditions. The most challenging aspect of the protocol is to precisely stimulate the fibular branch of the sciatic nerve, which innervates the anterior tibial muscles. To reduce inaccurate electrical stimulation, adjust the electrode position and stimulation amplitude until maximum twitch torque is recorded by a dynamometer.To begin, provide thermal support to the mouse by using an isothermal gel heating pad and place a heat lamp 1 meter above the mouse. To prepare the skin for DART or ISOM, remove the fur from the left hind limb by applying a depilatory cream. After 2 minutes, clean the leg with wipes soaked in distilled water to remove fur and all residual cream from the skin.Disinfect the skin using a povidone iodine scrubbing solution and 70%ethanol, then use a clean cotton swab to apply a protectant over the eyes and the depilated skin to prevent drying. Next, apply 5%lidocaine cream over the tibia to numb the area. Pass a 1/2-inch 26 gauge sterile hypodermic needle through the widest part of the proximal portion of the tibial bone.Once the stabilizing pin is secured, hold the needle with a sterile hemostat and bend the plastic portion until it breaks off, then lay the mouse in a supine position. Ensure that the mouse is still securely connected to the nose cone to maintain anesthesia. With a pair of sterile tipped tweezers, feed the tibial pin into a metal alligator clip, such that the end of the tibial pin are held by the alligator clamp.Move the adjustable arm of the alligator clamp to ensure that the foot of the mouse is placed on the footplate of the DART device. Strap the foot of the mouse onto the DART device footplate with adhesive laboratory tape. Ensure that the foot is placed at a 90-degree angle in relation to the long axis of the mouse tibial bone.Place an 18 gauge 1.5=inch-long hypodermic needle through the pre-drilled holes on the protractor of the DART device to create a plantarflexion stop, then rest the footplate on the plantarflexion stop. To optimize the electrode placement, place a bipolar, transcutaneous, and NMES electrode on the inferolateral aspect of the mouse knee joint. Use a laboratory electrical stimulator to apply single pulses of 1 hertz to stimulate the fibular branch of the sciatic nerve.Observe the tibialis anterior, or TA belly muscle and tendon for evidence of electrically-elicited twitch contractions. Now, strap the suture to the dynamometer floor plate, then optimize the amplitude of the voltage output from the NMES stimulator so that NMES is confined to the common fibular nerve and TA muscle. To set the stimulator to produce repeated pulse trains, adjust the dials for pulse frequency to 125 hertz, train duration to 500 milliseconds, and trains per second to 1.Turn on the toggle switch for repeating pulse trains. Set the stimulator to produce pulse trains that are 500 milliseconds in duration interspersed with 500 millisecond rest between pulse trains. Move the plantarflexion stop to the hole on the protractor that corresponds to 160 degrees on the long axis of the tibia.In DART, for the TA muscle to work concentrically, apply resistance by hanging a suitable weight such as 5 grams with a non-elastic silk suture tied to the DART device footplate. Adjust the resistance by applying 50%weight of the 1-repetition maximum. Ensure that the foot pulls through at least half of the available active range of dorsiflexion.Perform a single bout of DART training that involves 1 set of 10 repetitions of concentric contractions and 2-minute rest between the sets. For ISOM training, place the foot of the mouse at 160 degrees to the long axis of the tibia. Maintain the static position by taping the silk suture to the footplate of the robotic dynamometer.Perform a single bout of ISOM training that involves 4 sets of 10 repetitions to isometric contractions and 2-minute rest between sets. As post-procedural care for mice, take precautions to maintain the hygiene of the exercised hind limb and reduced needle site pain. Histological changes in the TA muscle were studied after 3 days of DART or ISOM training.Hematoxylin and eosin staining indicated that the extent of muscle damage was low in both DART and ISOM groups, but muscle damage was slightly more obvious in the ISOM group. It is important to specifically stimulate contractions in the anterior tibial muscles and precisely adjust the resistance against which these muscles have to work during dosage-adjusted resistance training. Following this procedure, investigators will be able to assess exercise tolerance in the form of susceptibility to fatigue and injury from repeated muscle contractions against resistance.This technique can be incorporated in a wide variety of basic and preclinical research applications, such as studies on mouse models, of neuromuscular diseases like muscular dystrophies and mouse models of sports injuries.

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