Name: dosage-adjusted resistance training in mice with a reduced risk of muscle damage
Uploaded: 2022-08-31T14:20:00.000+00:00
Duration: 7 min 29 s
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 authors have no competing financial interests.

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 the DART device34,35, the precise steps involved in performing DART or ISOM training, the proper harvesting and cryopreservation of muscle tissue, the proper sectioning of muscle tissue with a cryostat, and the proper staining of muscle cross-sections with hematoxylin and eosin22,36. Specifically, to construct the DART device, the parts must be fabricated with the exact dimensions and optimal material properties. If the dimensions are inaccurate for the wheel bearing housing, the 608-type wheel bearing will not fit snugly within the wheel bearing housing. If the dimensions of the mouse footplate and axle are not accurate, it might adversely impact the ability of the wheel bearing to move along with the mouse&#39;s foot. If the DART device parts are fabricated with an unsuitable material and/or 3D printer settings, the DART device parts might lack sufficient mechanical strength, which might lead to bending and/or breaking of various components34.
Modifications of this protocol might be needed based on the specific research questions that investigators wish to answer. The current protocol is specific to designing and implementing the DART device in a study that attempted to answer the question as to whether or not a single bout of DART causes extensive damage to the TA muscle in dysferlin-deficient mice, as we reported earlier with a similar bout of eccentric contractions22. Since others have suggested that exercise consisting of isometric contractions might be non-injurious and, therefore, suitable for humans with certain muscle diseases, we compared the extent of muscle damage caused by DART to a comparable bout of isometric contractions (ISOM)37,38. In this study, we found that both DART and ISOM induce minimal muscle damage, with DART showing slightly but significantly lower levels of damage than ISOM.
In relation to troubleshooting, the most challenging aspect of the protocol is precisely stimulating the fibular branch of the sciatic nerve, which gives motor innervation to the TA muscle. This technique is particularly challenging because the tester holds a transcutaneous electrode and places it manually on a precise spot that is inferior and lateral to the mouse&#39;s knee joint20,39. The tester must practice and learn how to locate this spot on the mouse&#39;s hindlimb by feeling for a slight bony prominence corresponding to the head of the mouse&#39;s fibular bone40. In order to confirm that optimal electrical stimulation of the fibular branch of the sciatic nerve is being achieved, such that maximal contractions from the TA muscle are achieved, it is best that a reliable dynamometery system is used20,21,22,41. Furthermore, transcutaneous or subcutaneous electrodes stabilized by a clamp may also be considered for reliable and reproducible placement of electrodes to minimize user-induced variability and errors20,41,42,43.
The main limitation of the protocol is that it is specifically designed to study the effect of DART on the TA muscle in mice. With methods that have been developed to perform dynamometric assessments and forced exercise on the quadriceps femoris muscle group in rodents, the DART device can easily be adapted for the quadriceps femoris muscle group42,43. Applying the DART device to other muscle groups might be more challenging; however, the cable-pulley-weight circuit design, which has been used in the DART device, can be incorporated into devices that are suitable for other muscle groups. Another limitation is that the protocol is performed under general anesthesia, making exercise forced and not voluntary; this is different from most resistance training paradigms developed for humans12,21.
The significance of the DART device and methodology with respect to existing or alternative methods is that the dosage for resistance training can be precisely adjusted and the exercise can be precisely targeted to a particular muscle group12. Precision rehabilitation is a new strategic priority for the United States National Institutes of Health, and, since DART makes it possible to perform precision resistance training in mice, DART lends itself well to basic and preclinical studies on precision physical rehabilitation44,45.
The importance and potential application of the current method of performing dosage-adjusted resistance training are that it makes it possible to perform resistance training studies in mice in ways comparable to human testing and training protocols used in clinical rehabilitation research and practice. For example, just as the one-repetition maximum (1RM, the maximum load that can be successfully lifted/moved/pressed/squatted just one time while maintaining good form) is used for humans to adjust the magnitude of resistance for training bouts17,18, the maximum load that the TA muscle can successfully lift can be used to set the resistance for training in mice with the DART device. In addition to adjusting the resistance based on an animal&#39;s capacity, the additional advantage is that the contractions are concentrically biased, which helps reduce contraction-induced muscle injury22. The representative results suggest that one bout of DART is even less injurious than a comparable bout of isometric contractions (ISOM group). The non-injurious nature of DART makes it appropriate for training studies where injurious contractions are best avoided&#160;&#8212;&#160;e.g., training studies in mice that model muscular dystrophies and training studies designed to gradually reload muscle following experimental surgical procedures on muscles and/or tendons22,46,47.

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.

<table><tbody><tr><td>AnMiao Star 608 Ceramic Ball Bearing</td><td>Anmiao Star (N/A)</td><td>AMS127</td><td>High precision, low friction wheel bearing.&amp;nbsp; If make and model is not commercially available, an alternative version of a 608 low-friction wheel bearing, 8 mm bore diameter,&amp;nbsp; 22 mm outside diameter, with silicon nitride ceramic balls in 420 stainless steel housing should suffice.&amp;nbsp; Excess friction in the wheel bearing will adversely impact performance of the DART device and will increase overall resistance to muscle contractions.</td></tr><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></tr><tr><td>B6.A-Dysfprmd/GeneJ (a.k.a. BLAJ mice)</td><td>The Jackson Laboratory (Bar Harbor, ME).&amp;nbsp; 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></tr><tr><td>Bipolar, transcutaneous, neuromuscular electrical stimulation (NMES) electrode</td><td>Harvard Apparatus, Holliston, MA</td><td>BS4 50&amp;ndash;6824</td><td>Electrode for NMES.&amp;nbsp; If this electrode is not commercially available, please contact corresponding author for alternatives.</td></tr><tr><td>Coplin Staining Dish</td><td>ThermoFisher (Waltham, MA)</td><td>Catalog No. S17495</td><td>Staining dish/jar for hematoxylin and eosin (H&amp;amp;E) staining of sections</td></tr><tr><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></tr><tr><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></tr><tr><td>Eosin Y</td><td>Millipore Sigma (Burlington, MA)</td><td>HT110132-1L</td><td>Pink cytoplasmic stain</td></tr><tr><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></tr><tr><td>Hematoxylin solution, Gill No.3</td><td>Millipore Sigma (Burlington, MA)</td><td>GHS332-1L</td><td>Dark blue stain for nuclei</td></tr><tr><td>HM525NX cryostat</td><td>ThermoFisher (Waltham, MA)</td><td>Catalog #HM525NX</td><td>Cryostat to make frozen sections of muscle</td></tr><tr><td>Lab Wipes.&amp;nbsp; Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply</td><td>ThermoFisher (Waltham, MA)</td><td>Catalog No. 06-666.&amp;nbsp; Manufacturer #34120</td><td>Laboratory wipes to blot mineral oil from muscle tissue before snap freezing and for other purposes.</td></tr><tr><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></tr><tr><td>Liquid nitrogen HDPE Dewar Flasks</td><td>ThermoFisher (Waltham, MA)</td><td>S34074B.&amp;nbsp; Thermo Scientific 41502000/EMD</td><td>Flask to hold liquid nitrogen for snap freezing muscle or other tissue</td></tr><tr><td>Magic depilatory cream</td><td>Softsheen Carson (New York, NY)</td><td>N/A</td><td>Razorless hair removal cream</td></tr><tr><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></tr><tr><td>Mineral Oil</td><td>ThermoFisher (Waltham, MA)</td><td>BP26291</td><td>Mineral oil to cryoprotect muscle tissue before snap freezing</td></tr><tr><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.&amp;nbsp; This is the material used to 3D print device components.</td></tr><tr><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></tr><tr><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.&amp;nbsp; NIH Image is also known as Image J.</td></tr><tr><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.&amp;nbsp; Stimulators, such as Model 4100 from A-M come with a built in stimulation isoloation unit</td></tr><tr><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 .&amp;nbsp; If this stimulator is not commercially available, Model 4100 Isolated High Power Stimulator from A-M systems could be an alternative.&amp;nbsp; Please contact co-author Jones for more information.</td></tr><tr><td>Scott&amp;rsquo;s bluing reagent</td><td>Ricca Chemical Company (Arlington, TX)</td><td>6697-32</td><td>Bluing solution that intensifies hematoxylin nuclear staining</td></tr><tr><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></tr><tr><td>Tabletop isoflurane vaporizer</td><td>VetEquip (Livermore, CA)</td><td>Item #901801</td><td>Inhaled tabletop anesthesia system</td></tr><tr><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></tr></tbody></table>

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. If it were found that DART was non-injurious to BLAJ mice, then it would likely be useful as a form of non-injurious resistance training, which could be applied alone or as an adjunct to regenerative medicine, genetic, pharmacological, and other interventions.
The ages and weights of BLAJ mice were closely matched between the DART and ISOM groups (Table 1). On Day 3 (~72 h), after a single bout of training, the exercised TA muscle had low levels of damage in both the DART and ISOM groups (&#60;10% damaged area)&#160;&#8212; this is in contrast to past studies21,22 of the response of BLAJ mice to eccentric muscle contractions, where ~40% damaged fibers have been reported at Day 3 (Figure 2, Table 2). When the area of muscle damage was compared between exercised TA muscles from the DART and ISOM groups, it was found that the DART group had lower levels of muscle damage than the ISOM group (Figure 2, Table 2). The maximum tetanic torque recorded on Day 0 (baseline) and Day 3 was not statistically different between the DART and ISOM groups (Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64000/64000fig01.jpg" /> 
Figure 1: Fabricating the DART device and applying it in a training study. (A,B) The DART device is based on a cable-pulley-weight circuit design, which is common to resistance training equipment that is designed for humans. (A) The DART device with an animal during a DART training session. (B) The footplate moving into dorsiflexion during a concentric contraction of the TA muscle (curved green arrow, right). The concentric contraction causes the 5 g resistance to move vertically against gravity (vertical green arrow, left). Muscle contractions were elicited with electrical stimulation applied through a transcutaneous bipolar electrode. (C) Various components of the DART device were designed with stereolithography software to generate .STL files, which could be opened with slicing software. With slicing software, G-CODE files were generated specific to the 3D printer and filament used. The 3D printed components of the DART device included (C) housing for a 608 low friction wheel bearing, (D) a tower for the wheel bearing housing, (E) a footplate, and (F) an axle to connect the footplate to the wheel bearing. The 3D printed components were combined and mounted on an acrylic base with glue and screw fasteners as described in the text and shown in (A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/64000fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64000/64000fig02.jpg" /> 
Figure 2: Histological study. Histological changes in the TA muscle at Day 3 (A) post-DART or (B) post-ISOM. Cryosections, which were 5 &#181;m in thickness, were stained with hematoxylin and eosin. Multiple overlapping digital images were captured and merged together with imaging software to generate high-resolution tiled images of the entire TA muscle cross-section. The qualitative histological data 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. The yellow arrows point to some of the damaged regions in TA muscle cross-sections. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/64000fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Ages and body weights of mice. The BLAJ mice that were studied were closely matched in age and body weight with no significant difference between the DART and ISOM groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/64000_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Quantitative analysis of TA muscle damage. The extent of muscle damage was expressed as a percentage of the total area of the TA muscle cross-section and analyzed by a T-test. Both DART and ISOM training resulted in a low level of muscle damage at Day 3 when compared to past studies involving a similar bout of eccentric contractions in BLAJ mice. Though the magnitude of muscle damage was small in both the DART and ISOM groups, the extent of damage was statistically lower in the DART group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/64000_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Contractile torque data. Contractile torque produced by the dorsiflexor muscles was studied with a robotic dynamometer connected to the DART device. There was no significant difference between the DART and ISOM groups in maximum baseline tetanic torque measured on the day of exercise (A, Day 0) or at 3 days post exercise (B, Day 3). Despite the lack of histological evidence of widespread muscle damage, a single bout of DART and ISOM was associated with a contractile torque deficit (~40%) on Day 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/64000_Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Video 1: DART training in mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/DART Concentric JoVE 031422.mp4" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2: ISOM training in mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/ISOM JoVE 031422.mp4" target="_blank">Please click here to download this Video.</a>
Supplementary File 1: Weight to torque calibration data, curve, and setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/Supplementary Data_070122.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Coding Files 1-4: Designs for the DART device components. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64000/Supplementary Coding Files 1-4.zip" target="_blank">Please click here to download this File.</a>

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 ...

dosage-adjusted-resistance-training-mice-with-reduced-risk-muscle

Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.7K Views.  Wayne State University. Dit protocol is een van de eerste studies die wetenschappers in staat stelt om doseringsgecorrigeerde weerstandstraining bij muizen uit te voeren, vergelijkbaar met hoe het bij mensen wordt uitgevoerd. Het grote voordeel van deze techniek is dat het onderzoekers in staat stelt om de weerstand nauwkeurig aan te passen waartegen de voorste tibiale spieren van de muis moeten werken tijdens weerstandstraining. Bovendien, omdat spiercontracties excentrisch bevooroordeeld zijn, is er een lagere kan...

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

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Behavior

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Medicine

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery.
The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. ...

Cancer Research

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s).
The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Electrocardiogram (ECG) is the key variable to understanding cardiac electrophysiology. Physical exercise has beneficial effects but may also be harmful in the context of cardiovascular diseases. This manuscript provides a method of recording real-time ECG during exercise, which can serve to investigate its effects on cardiac electrophysiology in mice.

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. ...

Exploring T2DM Exercise Interventions: A Static Training Device for Mice

Static Strength Training Method for Type 2 Diabetic Mice

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. Static strength training is one of the key forms of traditional sports in China. Research shows that static strength training is an effective clinical method for T2DM intervention, but there is no experimental device suitable for static training in mice. One of the difficulties in moving from clinical to basic research is to design appropriate experimental devices. In order to further study the mechanism of static training intervention in T2DM, a simple method for making a static training device for mice is introduced in this paper. This device has the advantages of simple operation, cheap material, and high feasibility. Previous studies conducted under this protocol have shown that static training can effectively reduce blood glucose levels and improve the mitochondrial function of skeletal muscle cells in T2DM mice. The purpose of introducing this device is to promote research on the mechanism of traditional exercise in the intervention of T2DM and to lay a foundation for the quantitative intervention of exercise.

Author Spotlight: Advancing Diabetes Research with Static Exercise Training in Mice

This protocol provides a simple method of making static training equipment for mice. The device maintains the muscle isometric contraction of the limbs of mice so as to verify the intervention effect of traditional exercise on type 2 diabetes (T2DM) and provides new exercise therapy for the clinical treatment of T2DM.

The treatment of type 2 diabetes mellitus (T2DM) is a major difficulty in improving patient health. Exercise is one of the main interventions for T2DM. ...

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

Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

The present protocol describes resistance training and testing using static and dynamic ladders in animal models.

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video