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>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+and+functional+effects+of+beta-hydroxy-beta-methylbutyrate+(HMB)+supplementation+in+skeletal+muscle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+strength+as+a+predictor+of+all-cause+mortality+in+healthy+men.">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. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+oxidative+stress+by+resveratrol+after+isometric+contractions+in+gastrocnemius+muscles+of+aged+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Assessment+of+Muscle+Contractility+in+Animal+Studies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+In+Vivo+Functional+Testing+of+the+Rat+Tibialis+Anterior+for+Evaluating+Tissue+Engineered+Skeletal+Muscle+Repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+muscle+function+of+the+extensor+digitorum+longus+muscle+ex+vivo+and+tibialis+anterior+muscle+in+situ+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isometric+and+eccentric+force+generation+assessment+of+skeletal+muscles+isolated+from+murine+models+of+muscular+dystrophies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+rodent+model+of+contraction-induced+injury+and+non-invasive+monitoring+of+recovery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+estrogen+receptor+alpha+in+skeletal+muscle+results+in+impaired+contractility+in+female+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+fast+and+slow+skeletal+muscles+from+mdx+mice+6-28+months+old.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Electrical+Stimulation+for+the+Assessment+of+Skeletal+Muscle+Contractile+Function+in+Murine+Models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+skeletal+muscle+atrophy.">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 border="0" cellpadding="0" cellspacing="0" style="width:606px;" width="605">
	<tbody>
		<tr height="229">
			<td height="229" style="height:229px;width:223px;">1300A: 3-in-1 Whole Animal System &#8211; Mouse</td>
			<td style="width:89px;">Aurora Scientific Inc.</td>
			<td style="width:123px;">305C-LR: Dual-Mode Footplate; 605A: Dynamic Muscle Data Acquisition And Analysis System; 701C: Electrical Stimulator and 809C:&#160;in-situ&#160;Mouse Apparatus</td>
			<td style="width:172px;">Complete muscle function system&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Conductive gel&#160;</td>
			<td style="width:89px;">Livingstone</td>
			<td style="width:123px;">ECGEL250</td>
			<td style="width:172px;">conductive gel used in the mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Eye ointment</td>
			<td style="width:89px;">Alcon</td>
			<td style="width:123px;">Poly Visc</td>
			<td style="width:172px;">pharmaceutic product (ophthalmic use)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">nonsteroidal anti-inflammatory drug (NSAID)&#160;</td>
			<td style="width:89px;">Ilium</td>
			<td style="width:123px;">Metacam</td>
			<td>veterinary medicine (injectable 5mg/ml)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Isoflurane&#160;</td>
			<td style="width:89px;">Zoetis</td>
			<td style="width:123px;">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/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/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/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/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/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

Nanyang Technological University

Research

JoVE Journal

Medicine

10.5K 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

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...