Funding from this project was from the School of Exercise and Nutrition Sciences, Deakin University. The authors would like to thank Mr. Andrew Howarth for his extensive work in optimizing the electrodes device.

All experimental procedures were approved by Deakin University Animal Ethics Committee (Project #G19/2014).
1. Equipment Setup
<ol>
	<li>Ensure that all machines are properly connected.</li>
	<li>Turn on the computer, the high-power bi-phase stimulator, and dual-mode lever system.</li>
	<li>Set up the mouse knee clamp on the platform, as well as the mouse footplate on the transductor.</li>
	<li>Turn on the heating platform to 37 &#176;C.</li>
	<li>Open the dynamic muscle control software on the desktop. 
	NOTE: This is the software needed to perform functional testing.</li>
</ol>
2. Software and Model Setup
<ol>
	<li>Once the program is opened (Figure 1), calibrate the transducer and select Setup | My Instruments | Calibrate.</li>
	<li>On the &#34;Setup&#34; button, select InstantStim and change the &#34;Run Time&#34; parameters to 120 s (Figure 1A). 
	NOTE: Optimal voltage can also be achieved by performing single twitches, manually setting up, or starting the InstantStim as many times as needed.</li>
	<li>In the type-able window labeled &#34;Autosave Base&#34; to input the name of the auto save file location (e.g., mouse1-date-timepoint1). Click the checkbox to the left of the &#34;Autosave Base&#34; window and change it to Enable Autosave.</li>
	<li>At the top of the DMC control screen go to Sequencer, which will open a new pop up window. Select Open Sequence and select the premade protocol to be used (Figure 1B). Click Load Sequence | Close Window. 
	NOTE: This step is used to generate a force frequency-curve (FFC) test (1, 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250 Hz).</li>
	<li>Set the &#34;RANGE&#34; knob to 10 mA on the bi-phase stimulator. 
	NOTE: Ensure that the &#34;ADJUST&#34; knob (right next bottom) is on zero. This fine adjustment allows the setup of the electrodes.</li>
</ol>
3. Mouse Setup
NOTE: All force measurements were performed on male wild-type mice (C57BI/6) at 12 weeks of age.
<ol>
	<li>Place each mouse into the anesthesia chamber with an oxygen flow rate of 1 L/min with 5% isoflurane (via nosecone inhalation) until the mouse loses consciousness. Confirm adequate anesthesia via loss of the foot reflex.</li>
	<li>Remove all hair on the right leg of the mouse by shaving with electric hair clippers.</li>
	<li>Place the animal in a supine position on the heated platform and clean the right leg (either side can be used) with 70% alcohol and iodine. At this point, adjust the isoflurane to 2% (with oxygen flow at 1 L/min) and apply the conductive gel to the skin where electrodes will be placed. 
	NOTE: Use a rectal temperature probe to monitor the body temperature during the procedure and apply eye ointment to prevent any dryness and/or damage to the eye.</li>
	<li>Place the foot onto the footplate and attach using medical tape. Clamp the knee to stabilize and immobilize the leg during the procedure. 
	NOTE: Some studies have described using a very thin pin inserted through the proximal tibia (posterior to the dorsiflexors muscles)12 to provide stabilization. This protocol opts for a clamp, as this provides sufficient stabilization without unnecessary compression/damage to the knee. The clamp also avoids potential inflammation that a trans-osseous pin might create, while still allowing accurate assessment of muscle contractility. Furthermore, the mouse knee clamp has been successfully used14.</li>
	<li>At this point, use the knobs on the platform to position the mouse hindlimb so that there is a 90&#176; angle at the ankle (Figure 2).</li>
</ol>
4. Optimization of the Electrodes Position
<ol>
	<li>Once the mouse is placed on the platform, position the electrodes under the skin (subcutaneous) in the right leg. 
	NOTE: This is a crucial step, and some repositioning may be required to get the desired position during the setup in step 4.4.</li>
	<li>Place the electrodes on the lateral side of the right leg; place one near the head of the fibula and the other electrode more distally on the lateral side of the leg (Figure 2). 
	NOTE: A custom-made electrode system is designed to optimize this step. However, this test can be performed with electrode needles provided by the manufacturer in this system.</li>
	<li>Once these steps are achieved, on the high-power bi-phase stimulator adjust the knob labeled &#34;ADJUST&#34; as needed to obtain a stimulation of the peroneal nerve that results in maximum dorsiflexion torque. 
	NOTE: For adult wild-type mice, this range is less than 2 mA; however, this may be dependent on the size, age, and sex of the animal. The force production (peaks of curves) should be increased slowly until the maximum force is reached.</li>
	<li>During stimulation, turn the transducer clockwise to yield negative values (Figure 3), which are important to ensure that the electrodes are stimulating only the dorsiflexor muscles by peroneal nerve. Once this step is achieved, stabilize the electrodes using a clamp, preventing any movement during the procedure. 
	NOTE: The peaks will slowly increase in magnitude, and the maximum amperage is determined as the level at which three or more consecutive stimulations result in identical contractility. Resist turning the amperage higher than necessary; the maximum amperage will stimulate the neighboring and potentially antagonist muscles to contract, causing co-contraction, which can generate peaks of positive values.</li>
	<li>Stop the Instant Stim on the software.</li>
	<li>On the main screen, turn on the button labeled &#34;Start Sequence&#34; to start the previous setup sequence (as described in step 2.4).</li>
</ol>
5. Ending the Procedure
<ol>
	<li>Once the force measurements are finished, remove the electrodes, release the knee clamp, and remove the foot tape.</li>
	<li>Turn off the isoflurane and maintain oxygen delivery for a few minutes aiding the animal recovery. Once the mouse starts moving and/or regains consciousness and can self-right, return the mouse to its cage. 
	NOTE: A nonsteroidal anti-inflammatory drug (NSAID) can be injected subcutaneously (1 mg/kg meloxicam) to prevent any discomfort and/or soreness after the procedure.</li>
</ol>
6. Data Analysis
<ol>
	<li>Open the data analysis software.</li>
	<li>Go to High Throughput (top-left on the screen). Select Force Frequency to analyze the above described setup sequence.</li>
	<li>Select Manual and change the &#34;End Cursor&#34; value to 3. Also select Remove Baseline.</li>
	<li>Click on Pick Files to access the previously performed procedure and then click Analyze. At this point the result can be accessed on the screen or exported to a spreadsheet for further analysis and/or calculations. 
	NOTE: The data were measured in mN; however, the torque can be calculated by multiplying the force value by the length of the lever arm (absolute force). If normalization is required (specific force), torque can be normalized to body weight, or terminal experiments can be performed to collect muscle mass of age-matched.</li>
</ol>

<ol><li>Frontera, W. R., Ochala, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle%3A+a+brief+review+of+structure+and+function%5BTitle%5D)+AND+%22Calcified+Tissue+International%22%5BJournal%5D)">Skeletal muscle: a brief review of structure and function.</a> Calcified Tissue International. 96 (3), 183-195 (2015).</li>
<li>Gerlinger-Romero, F., Guimaraes-Ferreira, L., Yonamine, C. Y., Salgueiro, R. B., Nunes, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+beta-hydroxy-beta-methylbutyrate+%28HMB%29+on+the+expression+of+ubiquitin+ligases%2C+protein+synthesis+pathways+and+contractile+function+in+extensor+digitorum+longus+%28EDL%29+of+fed+and+fasting+rats%5BTitle%5D)+AND+%22The+Journal+of+Physiological+Sciences%22%5BJournal%5D)">Effects of beta-hydroxy-beta-methylbutyrate (HMB) on the expression of ubiquitin ligases, protein synthesis pathways and contractile function in extensor digitorum longus (EDL) of fed and fasting rats.</a> The Journal of Physiological Sciences. 68 (2), 165-174 (2018).</li>
<li>Pinheiro, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+and+functional+effects+of+beta-hydroxy-beta-methylbutyrate+%28HMB%29+supplementation+in+skeletal+muscle%5BTitle%5D)+AND+%22European+Journal+of+Applied+Physiology%22%5BJournal%5D)">Metabolic and functional effects of beta-hydroxy-beta-methylbutyrate (HMB) supplementation in skeletal muscle.</a> European Journal of Applied Physiology. 112 (7), 2531-2537 (2012).</li>
<li>Metter, E. J., Talbot, L. A., Schrager, M., Conwit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+strength+as+a+predictor+of+all-cause+mortality+in+healthy+men%5BTitle%5D)+AND+%22The+Journal+of+Gerontology%2C+Series+A%3A+Biological+Sciences+and+Medical+Sciences%22%5BJournal%5D)">Skeletal muscle strength as a predictor of all-cause mortality in healthy men.</a> The Journal of Gerontology, Series A: Biological Sciences and Medical Sciences. 57 (10), B359-B365 (2002).</li>
<li>Foletta, V. C., White, L. J., Larsen, A. E., Leger, B., Russell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+and+regulation+of+MAFbx%2Fatrogin-1+and+MuRF1+in+skeletal+muscle+atrophy%5BTitle%5D)+AND+%22Pfl%C3%BCgers+Archiv%3A+European+Journal+of+Physiology%22%5BJournal%5D)">The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy.</a> Pflügers Archiv: European Journal of Physiology. 461 (3), 325-335 (2011).</li>
<li>Zacharewicz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+microRNAs+linked+to+regulators+of+muscle+protein+synthesis+and+regeneration+in+young+and+old+skeletal+muscle%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Identification of microRNAs linked to regulators of muscle protein synthesis and regeneration in young and old skeletal muscle.</a> PLoS One. 9 (12), e114009(2014).</li>
<li>Ryan, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+oxidative+stress+by+resveratrol+after+isometric+contractions+in+gastrocnemius+muscles+of+aged+mice%5BTitle%5D)+AND+%22The+Journal+of+Gerontology%2C+Series+A%3A+Biological+Sciences+and+Medical+Sciences%22%5BJournal%5D)">Suppression of oxidative stress by resveratrol after isometric contractions in gastrocnemius muscles of aged mice.</a> The Journal of Gerontology, Series A: Biological Sciences and Medical Sciences. 65 (8), 815-831 (2010).</li>
<li>Iyer, S. R., Valencia, A. P., Hernandez-Ochoa, E. O., Lovering, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Assessment+of+Muscle+Contractility+in+Animal+Studies%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">In Vivo Assessment of Muscle Contractility in Animal Studies.</a> Methods in Molecular Biology. 1460, 293-307 (2016).</li>
<li>Mintz, E. L., Passipieri, J. A., Lovell, D. Y., Christ, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+In+Vivo+Functional+Testing+of+the+Rat+Tibialis+Anterior+for+Evaluating+Tissue+Engineered+Skeletal+Muscle+Repair%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair.</a> Journal of Visualized Experiments. (116), e54487(2016).</li>
<li>Hakim, C. H., Wasala, N. B., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+muscle+function+of+the+extensor+digitorum+longus+muscle+ex+vivo+and+tibialis+anterior+muscle+in+situ+in+mice%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Evaluation of muscle function of the extensor digitorum longus muscle ex vivo and tibialis anterior muscle in situ in mice.</a> Journal of Visualized Experiments. (72), e50183(2013).</li>
<li>Moorwood, C., Liu, M., Tian, Z., Barton, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isometric+and+eccentric+force+generation+assessment+of+skeletal+muscles+isolated+from+murine+models+of+muscular+dystrophies%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isometric and eccentric force generation assessment of skeletal muscles isolated from murine models of muscular dystrophies.</a> Journal of Visualized Experiments. 71, e50036(2013).</li>
<li>Lovering, R. M., Roche, J. A., Goodall, M. H., Clark, B. B., McMillan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vivo+rodent+model+of+contraction-induced+injury+and+non-invasive+monitoring+of+recovery%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">An in vivo rodent model of contraction-induced injury and non-invasive monitoring of recovery.</a> Journal of Visualized Experiments. (51), e50036(2011).</li>
<li>Corona, B. T., Ward, C. L., Baker, H. B., Walters, T. J., Christ, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantation+of+in+vitro+tissue+engineered+muscle+repair+constructs+and+bladder+acellular+matrices+partially+restore+in+vivo+skeletal+muscle+function+in+a+rat+model+of+volumetric+muscle+loss+injury%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">Implantation of in vitro tissue engineered muscle repair constructs and bladder acellular matrices partially restore in vivo skeletal muscle function in a rat model of volumetric muscle loss injury.</a> Tissue Engineering Part A. 20 (3-4), 705-715 (2014).</li>
<li>Collins, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+of+estrogen+receptor+alpha+in+skeletal+muscle+results+in+impaired+contractility+in+female+mice%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology+%281985%29%22%5BJournal%5D)">Deletion of estrogen receptor alpha in skeletal muscle results in impaired contractility in female mice.</a> Journal of Applied Physiology (1985). 124 (4), 980-992 (2018).</li>
<li>Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Force+and+power+output+of+fast+and+slow+skeletal+muscles+from+mdx+mice+6-28+months+old%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.</a> The Journal of Physiology. 535 (Pt 2), 591-600 (2001).</li>
<li>Vitzel, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Electrical+Stimulation+for+the+Assessment+of+Skeletal+Muscle+Contractile+Function+in+Murine+Models%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">In Vivo Electrical Stimulation for the Assessment of Skeletal Muscle Contractile Function in Murine Models.</a> Methods in Molecular Biology. 1735, 381-395 (2018).</li>
<li>Jackman, R. W., Kandarian, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+basis+of+skeletal+muscle+atrophy%5BTitle%5D)+AND+%22American+Journal+of+Physiology+Cell+Physiology%22%5BJournal%5D)">The molecular basis of skeletal muscle atrophy.</a> American Journal of Physiology Cell Physiology. 287 (4), C834-C843 (2004).</li>
</ol>

The authors have nothing to disclose.

Measurement of maximal muscle contractile function in an accurate and repeatable manner is critical to the progressive assessment of genetic, metabolic, and muscle conditions17. Similarly, in vivo muscle contractile function allows for the assessment of novel treatments and therapeutics for debilitating muscle conditions. We demonstrate herein the measurement of force production of the dorsiflexor muscles of mouse lower hindlimb through an in vivo procedure.
Commercial apparatuses are efficient and helpful in performing this non-invasive procedure. This test provides important advantages related to the assessment of muscle contractile function while preserving a native physiological environment, in which blood supply and innervation remain intact. On the other hand, its disadvantages are related to normalization of the force per unit of cross sectional area of muscle (specific force), which can only be ascertained in an isolated muscle that is harvested after experimentation. However, the non-invasive test allows multiple measurements of contractile function of the flexor muscles in the same animal over time, resulting in reduced numbers of experimental animals being required, especially if the goal is to assess relative changes (changes in absolute force over time).
There are important steps that must be considered during this procedure in order to achieve consistent data over the timepoints. First, one should attempt to standardize animal positioning whenever possible. Second, during the setting up it is important to be consistent with electrode positioning so that optimal stimulation can be reached via stimulation of the peroneal nerve. The location of the electrodes should be on the lateral side of the (in this case right) leg, near to the head of the fibula and other further down the lateral side of the leg (Figure 2). Based on this, the custom-made electrodes are designed as such that both can be placed at same position every time. However, sufficient stimulation can also be achieved using the electrode needles provided with the commercial apparatuses. Third, it is crucial to achieve negative peaks during the voltage setup by turning clockwise the transducer connected to the footplate. Correct positioning of the mouse leg electrodes with maximum voltage setup has shown to be a technique that can be performed on the same mouse over time.
The ability to assess and track muscle function at different timepoints on the same animal is an important assessment to characterize different muscle diseases as well as their progression. Furthermore, this measurement of muscle dorsiflexion in mice can be a tool to assess the efficacy of potential treatments in a native physiological environment, with minimum metabolic stress12. Thus, it provides a technique in assessing muscle disease, its progression and potential treatment.

Skeletal muscle is an important metabolic tissue that comprises approximately 40% of the total body weight. It plays a crucial role in the control of energy metabolism and homeostasis1. Skeletal muscle mass is maintained by a fine balance between the rates of protein synthesis and degradation1. Numerous disease conditions affect these processes in skeletal muscle, leading to a net loss in muscle mass (atrophy). These include, but are not limited to, cancer, AIDS, aging, fasting, and limb immobilization2,3. In the aging population, loss of strength is associated with a loss of muscle mass and is a predictor of all-case mortality4. In this context, assessment of muscle function provides an important measure when determining the efficacy of therapeutic strategies to combat and/or prevent skeletal muscle wasting and loss of function.
Researchers have used many different approaches and animal models to understand the molecular pathways of muscle atrophy5,6 and the implications of these mechanisms on muscle contractile function2,3,7. Therefore, correlating changes at the molecular level to differences in muscle function is imperative in understanding how molecular level changes can impact muscle functionality.
Skeletal muscle function, especially in small rodents, is typically performed using three well-described procedures8,9 to detect impaired force production and/or monitor disease progression. (1) Ex vivo; where muscle is removed from the animal and incubated in a Ringer&#39;s bath solution to assess the muscle function using field stimulation10. (2) In situ; where the proximal attachment of the muscle remains in the animal and the distal tendon is connected to a force transducer, allowing muscle function to be performed by direct nerve stimulation11. (3) In vivo; where electrodes are placed subcutaneously to obtain nerve-evoked muscle force production9,12. While these three procedures are used for different purposes, they each possess advantages and disadvantages. Therefore, it is important to select an appropriate method based on the aim of the study. The main limitation with ex vivo experiments is the removal of muscle from its normal environment and the use of field stimulation. The in situ method maintains a normal blood supply and uses stimulation through the nerve, but normal anatomy is altered and the nature of the experiment is terminal; thus, this makes follow-up muscle function measurements impossible. The in vivo method described here most closely mimics normal physiology in that the anatomy is undisturbed, the neuromuscular bundle remains intact, and the experiment is not terminal, allowing follow-up measures within the same animal over time8.
Here, we describe an in vivo procedure that allows multiple measurements of muscle function in the same animal over time. This procedure involves the assessment of muscles of the anterior crural compartment &#8212; including the tibialis anterior(TA), extensor digitorum longus (EDL), and extensor hallicus longus (EHL) muscles, responsible for dorsiflexion &#8212; in a non-invasive procedure by fibular (also known as peroneal) nerve stimulation. The TA provides most of the force for ankle dorsiflexion13, with only minimal contribution by the EDL and EHL that control movement of the toes. This non-terminal protocol ensures the preservation of nerve and blood supply. This allows for the investigation of disease evolution and treatment efficacy over time in the most physiological environment currently available in an animal model.

<table><tbody><tr><td>1300A: 3-in-1 Whole Animal System &amp;ndash; Mouse</td><td>Aurora Scientific Inc.</td><td>305C-LR: Dual-Mode Footplate; 605A: Dynamic Muscle Data Acquisition And Analysis System; 701C: Electrical Stimulator and 809C:&amp;nbsp;in-situ&amp;nbsp;Mouse Apparatus</td><td>Complete muscle function system&amp;nbsp;</td></tr><tr><td>Conductive gel&amp;nbsp;</td><td>Livingstone</td><td>ECGEL250</td><td>conductive gel used in the mice</td></tr><tr><td>Eye ointment</td><td>Alcon</td><td>Poly Visc</td><td>pharmaceutic product (ophthalmic use)</td></tr><tr><td>nonsteroidal anti-inflammatory drug (NSAID)&amp;nbsp;</td><td>Ilium</td><td>Metacam</td><td>veterinary medicine (injectable 5mg/ml)&amp;nbsp;</td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Zoetis</td><td>Isoflo</td><td>veterinary inhalation Anaesthetic</td></tr></tbody></table>

non-invasive assessment of dorsiflexor muscle function in mice

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

The force-frequency curve is a useful test in which muscles can be stimulated by lower and higher frequencies to distinguish suboptimal and optimal force responses15. The force at lower frequencies can stimulate a single twitch, activating fewer and smaller motor units, and at higher frequencies a stable peak is reached, where isolated twitches fused (tetanus), reaching maximum force through activating all motor units16. In the test presented, the tetanic curve starts at ~60 Hz, where the potentiation can be visualized (Figure 4A) and the maximum force is determined at ~150 Hz (Figure 4B), when the plateau is reached with a completed fused curve9,16.
Any variation of these results may indicate that the muscles are not being properly stimulated by the electrodes. Electrode placement is an important step in the preparation of this procedure, as the electrical stimulation must be correctly positioned to innervate the peroneal nerve and thus fully activate the muscles of dorsiflexion, which it supplies (TA, EDL, and EHL). Correct electrode positioning results in the generation of negative peaks (Figure 3) during this process, whereas misalignment of the electrodes or higher amperage can lead to the stimulation of surrounding muscles, causing co-contraction of the neighboring muscles and antagonist muscles, which in turn generates peaks of positive values.
Figure 5A shows representative force frequency-curve data from a mouse across time, where the procedure was repeated once a week until 5 timepoints were completed. These observations have shown consistent force production values throughout the timepoints and/or observations measured. This procedure has also shown to be consistent between mice measurements, as Figure 5B shows the representative area under the curve of the FFC stimulated over 5 different observations in 6 mice tested once a week.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58696/58696fig1.jpg" /> 
Figure 1: Software system.&#160;(A)&#160;Control software illustration of the steps for setting up the &#34;Instant Stim&#34; parameters. On the background photo, click Setup | Instant Stim. On the small popped up window (front photo), set up the parameters.&#160;(B)&#160;Illustration of the &#34;Sequencer&#34; setup view. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58696/58696fig1large.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/58696/58696fig2.jpg" /> 
Figure 2: Mouse setup. Overview of the position of the anesthetized animal. The right knee clamp is placed so that the knee is at 90&#176; and so that the foot and the ankle are at 90&#176; angles (dotted white line). Contraction of the dorsiflexors muscles is achieved by stimulation of the peroneal nerve, which is located just under (distal to) the head of the fibula. We use custom-designed electrodes (inset); however, needle electrodes that are provided with the unit, or purchased separately, are also sufficient. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58696/58696fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58696/58696fig3.jpg" /> 
Figure 3: Output from placement of the electrodes. Once the electrodes are positioned under the skin and the voltage is initiated, peaks with negative values are observed. At this point, reaching negative values (green lines) is a crucial step in making sure that the stimulation is achieved in the dorsiflexor muscles only (TA, EDL, and EHL). The real-time measurement is indicated between the two red lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58696/58696fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58696/58696fig4.jpg" /> 
Figure 4: Representative curves.&#160;(A)&#160;Sample of the force curve at 60 Hz (mouse #06). (B)&#160;Sample of the tetanic curve at 150 Hz (mouse #03). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58696/58696fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58696/58696fig5.jpg" /> 
Figure 5:&#160;Representative force frequency curve (FFC) and area under the curve data.&#160;(A)&#160;FFC (x-axis) over 5 different timepoints (weeks 1, 2, 3, 4, and 5) in a sample mouse (#05). (B)&#160;Area under the curve (AU, y-axis) of the FFC over 5 different timepoints (mouse #01, 02, 03, 04, 05, and 06, respectively; x-axis). Results are expressed as mean &#177; standard error of measurement (SEM) of five timepoints (tests) in 6 mice and were analyzed by one-way ANOVA test (p &#60; 0.05).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58696/58696fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Non-invasive Assessment of Dorsiflexor Muscle Function in Mice at JoVE.com

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

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

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

non-invasive-assessment-of-dorsiflexor-muscle-function-in-mice

Ateneo de Manila University

Research

JoVE Journal

Medicine

10.7K Views.  Deakin University. Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Video: Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

Behavior

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Neuroscience

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

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

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

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

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

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Biology

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

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

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

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

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

In vivo Measurement of Knee Extensor Muscle Function in Mice

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

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

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

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...

Terminal H-reflex Measurements in Mice

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits after injuries such as spinal cord damage or stroke. An increase of the H-reflex response, together with symptoms like non-voluntary muscle contractions, pathologically augmented stretch reflex, and hypertonia in the corresponding muscle, is an indicator of post-stroke spasticity (PSS).
In contrast to rather nerve-unspecific transcutaneous measurements, here, we present a protocol to quantify the H-reflex directly at the ulnar and median nerves of the forepaw, which is applicable, with minor modifications, to the tibial and sciatic nerve of the hindpaw. Based on the direct stimulation and the adaptation to different nerves, the method represents a reliable and versatile tool to validate electrophysiological changes in spasticity-related disease models.

The clinical evaluation of spasticity based on the Hoffmann reflex (H-reflex) and using electrical stimulation of peripheral nerves is an established method. Here, we provide a protocol for a terminal and direct nerve stimulation for H-reflex quantification in the mouse forepaw.

The Hoffmann reflex (H-reflex), as an electrical analog to the stretch reflex, allows electrophysiological validation of the integrity of neural circuits ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Summary

Explore More Videos

Assessing Functional Performance in the Mdx Mouse Model

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

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

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

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

In vivo Measurement of Knee Extensor Muscle Function in Mice

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Terminal H-reflex Measurements in Mice