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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#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>
		</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).&#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>
		</tr>
		<tr>
			<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>
		</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&#38;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.&#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>
		</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.&#160; 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.&#160; 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.&#160; 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.&#160; 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 .&#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>
		<tr>
			<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>
		</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. 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.

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

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Simulator Training for Endovascular Neurosurgery

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk environments. In the field of endovascular neurosurgery, the demand for consequence- and risk-free learning environments led to the development of simulation devices valuable for medical trainees. The goal of this protocol is to provide instructive guidelines for the use of an endovascular neurosurgery simulator in an academic setting. The simulator provides trainees with the opportunity to receive realistic feedback on their knowledge of anatomy, as well as haptic feedback indicative of their success in handling the catheter-based systems without negative consequences. The utility of this specific protocol in relation to other neuroendovascular training modalities is also discussed.

Simulation of complex, high-risk procedures is critical to the education of medical trainees. A protocol for simulator-based endovascular neurosurgery training in a controlled academic environment is described. The protocol includes stepwise guidelines for trainees of varying levels, with a discussion of the advantages and limitations of this model.

Simulation-based training has become common practice across medical specialties, especially for learning complex skills performed in high-risk ...

Assessment of Audio-Tactile Sensory Substitution Training in Participants with Profound Deafness Using the Event-Related Potential Technique

This paper examines the application of electroencephalogram-based methods to assess the effects of audio-tactile substitution training in young, profoundly deaf (PD) participants, with the aim of analyzing the neural mechanisms associated with vibrotactile complex sound discrimination. Electrical brain activity reflects dynamic neural changes, and the temporal precision of event-related potentials (ERPs) has proven to be key in studying time-locked processes while performing behavioral tasks that involve attention and working memory. 
The current protocol was designed to study electrophysiological activity in PD subjects while they performed a continuous performance task (CPT) using complex-sound stimuli, consisting of five different animal sounds delivered through a portable stimulator system worn on the right index finger. As a repeated-measures design, electroencephalogram (EEG) recordings in standard conditions were performed before and after a brief training program (five 1 h sessions over 15 days), followed by offline artifact correction and epoch averaging, to obtain individual and grand-mean waveforms. Behavioral results show a significant improvement in discrimination and a more robust P3-like centroparietal positive waveform for the target stimuli after training. In this protocol, ERPs contribute to the further understanding of learning-related neural changes in PD subjects associated with audio-tactile discrimination of complex sounds.

This protocol is designed to explore underlying learning-related electrophysiological changes in subjects with profound deafness after a short training period in audio-tactile sensory substitution by applying the event-related potential technique.

This paper examines the application of electroencephalogram-based methods to assess the effects of audio-tactile substitution training in young, ...

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...

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

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An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Laminectomy and Spinal Cord Window Implantation in the Mouse

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Simulator Training for Endovascular Neurosurgery

Assessment of Audio-Tactile Sensory Substitution Training in Participants with Profound Deafness Using the Event-Related Potential Technique

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice