This work was supported by grants from the National Institutes of Health (AR-49419, DD), Muscular Dystrophy Association (DD), and NIH training grant T90DK70105 (CH).

1. Evaluation of the Contractile and Passive Properties of the EDL Muscle Ex vivo
The contractile and passive properties of the EDL muscle are measured ex vivo using the Aurora Scientific in vitro muscle test system. Refer to Table 1 for materials and equipment.
1.1 Equipment preparation
<ol>
	<li>Assemble the tissue-organ bath by securing the oxytube to the water-jacket tissue bath. Attach the assembled bath to the muscle mounting apparatus. Connect the gas line to the oxytube. Fasten the water circulation lines to the water-jacket tissue bath and place the needle valve into the bath drainage.</li>
	<li>Turn on the circulating water-bath and adjust the temperature to 30 &deg;C 7. Allow 5 PSI (pounds per square inch) of 95% O2-5% CO2 to flow through the oxytube. Fill the bath with Ringer's buffer. Equilibrate the buffer for at least 10 min with a steady gas flow by adjusting the oxytube valve.</li>
	<li>Turn on the instruments (stimulator, dual-mode lever system, and signal interface). Load the dynamic muscle control (DMC) software according to manufacturer's instruction.</li>
</ol>
1.2 EDL muscle dissection
All animal studies must be approved by the Institutional Animal Care and Use Committee.
<ol>
	<li>Anesthetize the mouse with intraperitoneal injection of 2.5 &mu;l/g body weight of the anesthetic cocktail (refer to materials section). Throughout the surgical procedure, the depth of sedation was checked by performing a toe pinch. A supplement of 10% of the initial anesthetic dose is administrated when needed to keep the animal under anesthesia. Shave the hind limb. Maintain the body core temperature at 37 &deg;C prior to dissection procedure by placing the mouse on a heating pad. The body temperature is monitored by constantly measuring the rectal temperature using a thermal probe. </li>
	<li>Position the mouse supine on the dissection board (Figure 1). Peel off the leg skin to expose the hind limb muscles. Secure the leg on the sylgard block using two dressmaker pins, one in the foot and the other in the gracilis muscle. Place a heat lamp above the mouse body to maintain the core body temperature at 37 &deg;C. Constantly superfuse all exposed muscles with Ringer's buffer. Drain excess buffer through a vacuum line.</li>
	<li>Expose the distal TA tendon and the extensor ligament under a stereomicroscope by dissecting the skin toward the foot. Gently remove the fascia covering the TA muscle. Cut the extensor ligament to release the distal TA tendon.</li>
	<li>Cut the distal TA tendon and use it to peel off the TA muscle. Carefully remove the TA muscle at its proximal attachment. Place a thin piece of Ringer's buffer soaked cotton next to the EDL muscle to absorb bleeding caused by the rupture of the TA muscle vasculature. Use the vacuum line to remove excess buffer and blood.</li>
	<li>Tie a double square knots followed by a loop knot using a bread silk suture at the muscle tendon junction (MTJ) of the distal EDL muscle (Figure 2). Make an incision in the distal portion of the biceps femoris muscle to expose the proximal EDL muscle. Repeat the same set of knots (Figure 2) at the MTJ of the proximal EDL tendon. Attach the lever arm hook to either the proximal or the distal knots with a double square knot using the same suture line. Cut off the remaining suture line.</li>
	<li>Cut the proximal EDL tendon superior to the proximal suture knot. Lift up the EDL muscle with the hook and cut the vasculature beneath the muscle. Cut the distal EDL tendon inferior to the distal suture knot to remove the EDL muscle from the hind limb. Cover the exposed hind limb with a piece of Ringer's buffer soaked cotton.</li>
	<li>Attach the hook to the lever arm. Align the muscle vertically between two electrodes. Secure the distal suture line to the fixed post. Lift up the tissue bath to submerge the muscle in Ringer's buffer. Adjust the resting tension to 1.0 g using the dual coarse/fine translation stage and allow the muscle to equilibrate for at least 10 min.</li>
</ol>
1.3 Measuring the contractile and passive properties of the EDL muscle
Use Table 2 to set up the parameters in the DMC software for each of the following measurements. Analyze the data using the dynamic muscle analysis (DMA) software.
1.3.1 Measuring the contractile properties of the EDL muscle
<ol>
	<li>Stimulate the EDL muscle three times at 150 Hz with 60 sec apart to stabilize the muscle 8.</li>
	<li>Stimulate the EDL muscle at different resting tensions to determine the optimal length (Lo). The optimal length is the length at which muscle develops maximum twitch tension. Allow the muscle to relax for 2 min.</li>
	<li>Adjust the resting tension to Lo. Measure muscle force at single twitch stimulation. Determine the absolute twitch force (Pt), time to peak tension (TPT) and half relaxation time (&frac12; RT) of the Pt. Allow the muscle to relax for 2 min.</li>
	<li>Adjust the resting tension to Lo. Measure tetanic muscle force generated at different stimulation frequencies (50, 80, 100, 120, 150 and 200 Hz). Determine the absolute maximal tetanic muscle force (Po) where muscle force reaches the maximal. Measure the TPT and &frac12; RT of the Po 9.</li>
	<li>Allow the muscle to relax for 5 min. Adjust the resting tension to Lo. Apply 10 cycles of eccentric contractions with 2 min rest between cycles. Calculate the relative force loss of the Po after each cycle of eccentric contraction.</li>
	<li>Detach the EDL muscle from the apparatus and cut the tendons at the suture site. Determine the muscle wet weight and calculate the muscle cross sectional area (CSA) 6,10.</li>
</ol>
1.3.2 Measuring the passive properties of the EDL muscle
<ol>
	<li>Dissect the contralateral EDL muscle and attach it to the apparatus as described in Section 1.2, steps 2 to 7.</li>
	<li>Subject the EDL muscle to a six-step stretching protocol where the muscle is strained to 160% Lo with an increment of 10% Lo. Analyze the stress-strain profile 6.</li>
	<li>Evaluate the viscous property of the EDL muscle by measuring the stress relaxation rate (SRR) at the following time frames after stretching and holding the muscle at 10% Lo: from peak to 0.1s post-peak (pp), from 0.1 to 0.2s pp, from 0.2 to 0.5s pp, from 0.5 to 1s pp and from 1 to 1.5s pp.</li>
	<li>At the end of study, euthanize the mouse by cervical dislocation and/or decapitation while the mouse is still under anesthesia. Detach the EDL muscle from the apparatus and cut the tendons at the suture site. Determine the muscle wet weight and calculate the muscle cross sectional area (CSA) 6,10.</li>
</ol>
2. Evaluation of the Contractile Properties of the TA Muscle In situ
The contractile properties of the TA muscle are measured using the Aurora Scientific in situ muscle test system. Refer to Table 1 for materials and equipment.
2.1 Equipment preparation
<ol>
	<li>Heat up the thermo-controlled animal stage to 37 &deg;C using the circulating water-bath.</li>
	<li>Turn on the instruments (stimulator, dual-mode lever system, and signal interface). Load the DMC software according to manufacturer's instruction.</li>
</ol>
2.2 Preparation of the TA muscle for in situ force measurement
<ol>
	<li>Anesthetize the mouse, shave the hind limb and expose the TA muscle as described in steps 1 to 3 in Section 1.2.</li>
	<li>Tie a double square knot around the patella ligament using a bread silk suture. Tie a double square knot followed by a loop knot at the MTJ of the distal TA muscle (Figure 2), tie another double square knot leaving a ~10 mm loop from the distal TA tendon knot using the same suture line. Place the second double square knot on the side of the loop.</li>
	<li>Remove the pins from the hind limb and position the animal prone. Expose the biceps femoris muscle. Make an incision in the midline to reveal the sciatic nerve. Tie a double square knot around the proximal end of the sciatic nerve. Trim one side of the suture lines and cut the nerve superior to the knot. Gently, pull the sciatic nerve toward the knee using the suture line and clear the surrounding connective tissue to free ~ 5 mm of its length. Do not stretch the nerve during this procedure and constantly superfuse the nerve with ringer buffer.</li>
	<li>Prepare the contralateral TA muscle as described in steps 1 to 3. Cover one of the exposed hind limb with a piece of Ringer's buffer soaked cotton. Constantly superfuse both hind limbs with pre-warmed (37 &deg;C) Ringer's buffer. Remove excess buffer through a vacuum line.</li>
	<li>Position the animal prone on the animal platform. Attach the knee clamp holder to the animal platform and secure both knees to the metal pin with double square knots using the patella ligament suture lines. Pin both feet on the sylgard block using dressmaker pins. Secure the animal platform onto the thermo-controlled stage. Position the heat lamp to maintain the animal core body temperature at 37 &deg;C.</li>
	<li>Secure the electrode holder to the animal platform and lay the sciatic nerve on the electrode using the suture line. Keep the electrode away from the hind limb muscles. Cut the distal TA tendon of the uncovered hind limb at the MTJ suture site. Attach the distal TA tendon suture loop to the lever arm hook. Cover the exposed hind limb muscle with a warm Ringer's buffer soaked cotton.</li>
</ol>
2.3 Measuring the contractile properties of the TA muscle
<ol>
	<li>Use Table 2 to set the parameters in the DMC software. Follow the same protocol described in Section 1.3.1 to determine the contractile properties of the TA muscle. Analyze the data using the DMA software.</li>
	<li>After contractile property measurement, detach the distal TA tendon suture loop from the leveler arm hook. Remove the TA muscle. Determine the muscle wet weight and calculate the CSA 10.</li>
	<li>Measure the contractile properties of the contralateral TA muscle according to steps 1 to 3 described above. Euthanize the mouse according to institutional guidelines at the end of the study.</li>
</ol>

<ol>
	<li>Huijing, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+as+a+collagen+fiber+reinforced+composite:+a+review+of+force+transmission+in+muscle+and+whole+limb.">Muscle as a collagen fiber reinforced composite: a review of force transmission in muscle and whole limb.</a> J. Biomech. 32, 329-345 (1999).</li><li>Moss, R. L., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+and+viscous+properties+of+resting+frog+skeletal+muscle.">Elastic and viscous properties of resting frog skeletal muscle.</a> Biophys. J. 17, 213-228 (1977).</li><li>Hoffman, E. P., Brown, R. H., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin:+the+protein+product+of+the+Duchenne+muscular+dystrophy+locus.">Dystrophin: the protein product of the Duchenne muscular dystrophy locus.</a> Cell. 51, 919-928 (1987).</li><li>Petrof, B. J., Shrager, J. B., Stedman, H. H., Kelly, A. M., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+protects+the+sarcolemma+from+stresses+developed+during+muscle+contraction.">Dystrophin protects the sarcolemma from stresses developed during muscle contraction.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 3710-3714 (1993).</li><li>Pastoret, C., Sebille, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mdx+mice+show+progressive+weakness+and+muscle+deterioration+with+age.">mdx mice show progressive weakness and muscle deterioration with age.</a> J. Neurol. Sci. 129, 97-105 (1995).</li><li>Hakim, C. H., Grange, R. W., 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=The+passive+mechanical+properties+of+the+extensor+digitorum+longus+muscle+are+compromised+in+2-+to+20-mo-old+mdx+mice.">The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to 20-mo-old mdx mice.</a> J. Appl. Physiol. 110, 1656-1663 (2011).</li><li>Segal, S. S., 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=Temperature-dependent+physiological+stability+of+rat+skeletal+muscle+in+vitro.">Temperature-dependent physiological stability of rat skeletal muscle in vitro.</a> Am. J. Physiol. 248, 265-270 (1985).</li><li>Grange, R. W., Gainer, T. G., Marschner, K. M., Talmadge, R. J., Stull, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-twitch+skeletal+muscles+of+dystrophic+mouse+pups+are+resistant+to+injury+from+acute+mechanical+stress.">Fast-twitch skeletal muscles of dystrophic mouse pups are resistant to injury from acute mechanical stress.</a> Am. J. Physiol. Cell Physiol. 283, 1090-1101 (2002).</li><li>Hakim, C. H., 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=Gender+differences+in+contractile+and+passive+properties+of+mdx+extensor+digitorum+longus+muscle.">Gender differences in contractile and passive properties of mdx extensor digitorum longus muscle.</a> Muscle Nerve. 45, 250-256 (2012).</li><li>Hakim, C. H., Li, D., 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=Monitoring+murine+skeletal+muscle+function+for+muscle+gene+therapy.">Monitoring murine skeletal muscle function for muscle gene therapy.</a> Methods Mol. Biol. 709, 75-89 (2011).</li></ol>

Open access fees have been paid for by Aurora Scientific.

In this protocol, we have illustrated physiological assays for measuring the contractile and passive properties the EDL muscle and the contractile properties of the TA muscle. A major concern in muscle physiology studies is the oxygenation of the target muscle. For large muscles (such as the TA muscle), the in situ approach is preferred because oxygen diffusion from Ringer's buffer may not reach the center of the muscle in an in vitro assay. In situ approach does not disturb normal blood supply and hypoxia-associated artificial effects are avoided. The EDL muscle is one of the most commonly used muscle in physiology study. Adequate oxygenation of the entire muscle can be achieved in an in vitro system because of the small size of the muscle. Further, the in vitro system provides an enclosed environment to manipulate the concentrations of ions (Ca 2+, Na+ and K+) and chemicals (ATP and glucose) that are necessary for optimal muscle force generation. This offers a great opportunity to study the effect of these variables on force production.
Accurate measurement of the contractile and passive properties of the limb muscle is critical to study skeletal muscle function. Characteristic changes of these properties are often considered as the hallmarks of various muscle diseases. Changes in these parameters are also important indicators to determine whether an experimental therapy is effective or not.

<table><tbody><tr><td>Tissue-organ bath </td><td>Radnoti LLC, CA, USA</td><td></td><td>Water-jacket tissue bath (Cat #158351-LL), Oxygen disperser tube (Cat #160192), Luer valve (Cat#120722)</td></tr><tr><td>Circulating water bath </td><td>Fisher Scientific, Waltham, MA, USA</td><td></td><td></td></tr><tr><td>Gas mix </td><td>Airgas National, Charlotte, NC, USA</td><td></td><td>95% O&lt;sub&gt;2&lt;/sub&gt; and 5% CO&lt;sub&gt;2&lt;/sub&gt; </td></tr><tr><td>&lt;em&gt;In vitro&lt;/em&gt; muscle function assay apparatus </td><td><a href="http://www.aurorascientific.com/product/mp-model-1200/" target="_blank"><img src="/img/new_window.png" /> Aurora Scientific, Aurora, ON, Canada</a></td><td></td><td>The system consists of a stimulator (Model# 701A), a dual-mode lever system (Model#300C or 305C), a signal interface (Model # 604B) and a test apparatus (Model# 800A) to vertically mount tissue organ bath</td></tr><tr><td>&lt;em&gt;In vitro&lt;/em&gt; muscle function assay software</td><td><a href="http://www.aurorascientific.com/product/dmc-dma/" target="_blank"><img src="/img/new_window.png" /> Aurora Scientific, Aurora, ON, Canada</a></td><td></td><td>Dynamic muscle control (DMC) software and dynamic muscle control data analysis (DMA) software</td></tr><tr><td>Mouse anesthesia cocktail mixed in 0.9% NaCl</td><td>Refer to the institutional guidelines</td><td></td><td>Ketamine (25 mg/ml), xylazine (2.5 mg/ml) and acepromazine (0.5 mg/ml). Throughout the surgical procedure, a supplement of 10 % of the initial dose may be needed to keep animal under anesthesia. </td></tr><tr><td>Sylgard</td><td>World Precision Instrument</td><td>Cat#SYLG184</td><td></td></tr><tr><td>A custom-made Plexiglas dissection board</td><td>In house designed</td><td></td><td>Refer to &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>Heating lamp </td><td>Tensor Lighting Company, Boston, MA, USA</td><td></td><td>15 Watt lamp to keep the mouse warm during dissection</td></tr><tr><td>Ringer&#039;s Buffer </td><td>Chemicals are purchased from Fisher Scientific, Waltham, MA, USA</td><td></td><td>Composition in mM: 1.2 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (Cat#S369) , 1 MgSO&lt;sub&gt;4&lt;/sub&gt; (Cat# M63), 4.83 KCl (Cat# P217), 137 NaCl (Cat# 217), 24 NaHCO&lt;sub&gt;3&lt;/sub&gt; (Cat# S233), 2 CaCl&lt;sub&gt;2&lt;/sub&gt; (Cat #C79) and 10 glucose (Cat# D16). Dissolve chemicals individually and mix in the order listed above. Store at 4 &amp;deg;C.</td></tr><tr><td>Stereo dissecting microscope </td><td>Nikon, Melville, NY, USA</td><td></td><td></td></tr><tr><td>Dissection tools </td><td>Fine Science Tools, Foster City, CA, USA</td><td></td><td>Coarse forceps, coarse scissors, fine forceps (Straight and 45 &amp;deg; angle)</td></tr><tr><td>Braided silk suture #4-0 </td><td>SofSilk USSC Sutures, Norwalk, CT, USA</td><td>Cat # SP116</td><td></td></tr><tr><td>A custom-made stainless steel hook</td><td>Small Parts, Inc.</td><td></td><td>2&#039;&#039; long S/S 304V (0.18&#039;&#039; diameter) for force transducer 305C or 2.5&#039;&#039; long S/S 304V (0.012&#039;&#039; diameter) for transducer 300C (Cat# ASTM A313) </td></tr><tr><td>&lt;em&gt;In situ&lt;/em&gt; muscle function assay system </td><td><a href="http://www.aurorascientific.com/product/mp-model-1300/" target="_blank"><img src="/img/new_window.png" /> Aurora Scientific, Aurora, ON, Canada</a></td><td></td><td>The system (809B, &lt;em&gt;in situ&lt;/em&gt; mouse apparatus) consist of a stimulator (Model# 701B), a dual-mode lever system (Model# 305C), a signal interface (Model# 604A) and a thermo controlled footplate apparatus (Model# 809A)</td></tr><tr><td>&lt;em&gt;In vitro&lt;/em&gt; muscle function assay software</td><td><a href="http://www.aurorascientific.com/product/dmc-dma/" target="_blank"><img src="/img/new_window.png" /> Aurora Scientific, Aurora, ON, Canada</a></td><td></td><td>Dynamic muscle control (DMC) software and dynamic muscle control data analysis (DMA) software</td></tr><tr><td>A custom-made TA assay animal platform</td><td>In house designed</td><td></td><td>Refer to &lt;strong&gt;Figure 2&lt;/strong&gt;</td></tr><tr><td>A custom-made stainless steel hook</td><td>Small Parts, Inc.</td><td>Cat# ASTM A313</td><td>0.5&#039;&#039; long S/S 304V (0.18&#039;&#039; diameter) </td></tr><tr><td>Custom-made 25G platinum electrodes</td><td>Chalgren Enterprises, Gilroy,CA</td><td></td><td>Solder two 0.016&#039;&#039; thick platinum wires to two 24G electric wires</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Table 1.&lt;/strong&gt; Materials and equipment.&lt;/p&gt;</td></tr></tbody></table>

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.

The following results are a representation from our previous reports 6,9. Data are presented as mean&plusmn;standard error of mean. Table 3 shows the morphometric properties of the EDL muscle in normal BL10 and dystrophin-deficient (mdx) mice at 4 to 6 months of age. Figure 4 shows representative contractile and passive properties of the EDL muscle from BL10 and mdx mice. The contractile properties of the EDL muscle are described by the following terms including the specific (absolute force divided by the CSA) twitch force (Figure 4A), specific maximal tetanic force (Figure 4B), TPT and &frac12; RT of the absolute maximal tetanic force (Figure 4C and D). The TPT and &frac12; RT can also be calculate from the absolute twitch force. The stress-strain profile (Figure 4E) and SRR (Figure 4F) are used to describe the passive properties of the EDL muscle.
Absence of dystrophin has a significant impact on the contractile and passive properties of the EDL muscle 6,9. Specific twitch and tetanic forces are significantly reduced in the mdx EDL muscle. The TPT is significantly faster while the &frac12; RT is significantly slower in the mdx EDL muscle. The stress-strain profile suggests that stiffness is significantly increased in the mdx EDL muscle. The mdx EDL muscle also yields a significantly much higher resistance force (passive stress) before reaching the peak stress, while the post-peak stresses decline much faster. Further, the SRR was significantly higher in the mdx EDL muscle compared to that of the BL10 EDL muscle.
Statistical analysis
Statistical significance between two groups is analyzed by the Student t-test. For statistical significance among multiple groups, One-way or Two-way ANOVA analysis followed by Bonferroni post hoc analysis is recommended using the SAS software (SAS Institute Inc., Cary, NC). Difference is considered significant when p &lt; 0.05.
Table 1. Materials and equipment. 
<table border="1">
 <tbody>
 <tr>
 <td class="jove_content_small">Experiment</td>
 <td class="jove_content_small">Resting tension (gram)</td>
 <td class="jove_content_small">Pulse frequency (Hz) </td>
 <td class="jove_content_small">Pulse width (ms)</td>
 <td class="jove_content_small">Stimulation duration (ms)</td>
 <td class="jove_content_small">Stretch length </td>
 <td class="jove_content_small">Stretch duration (ms) </td>
 <td class="jove_content_small">Stretch rate </td>
 <td class="jove_content_small">Comments</td>
 </tr>
 <tr>
 <td colspan="9">1. Evaluation of contractile and passive properties of the EDL muscle ex vivo</td>
 </tr>
 <tr>
 <td colspan="9">1.3.1 Measuring the contractile properties of the EDL muscle </td>
 </tr>
 <tr>
 <td>1. Warm up</td>
 <td>1.0</td>
 <td>150</td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Rest the muscle for 60 sec between each stimulus. These preliminary tetanic contractions stabilize the muscle for subsequent measurements.</td>
 </tr>
 <tr>
 <td>2. Optimal muscle length (Lo)</td>
 <td>0.5, 1.0, 1.5 and 2.0</td>
 <td>1</td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Allow the muscle to relax for 30 sec between each stimulus. Measure the muscle optimal length using a digital caliper.</td>
 </tr>
 <tr>
 <td>3. Single twitch force (Pt)</td>
 <td>Adjust resting tension to Lo</td>
 <td>1</td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>4. Tetanic muscle force </td>
 <td>Adjust resting tension to Lo</td>
 <td>50, 80, 100, 120, 150 and 200 </td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Allow the muscle to relax for 1 min between each stimulus. Determine the frequency that generate the maximal absolute tetanic force (Po).</td>
 </tr>
 <tr>
 <td>5. Eccentric contraction</td>
 <td>Adjust resting tension to Lo</td>
 <td>Use the frequency that generates the maximum tetanic force (Po) </td>
 <td>0.2</td>
 <td>700</td>
 <td>10% Lo</td>
 <td>last 200 ms of the stimulation duration</td>
 <td>0.5 Lo/sec</td>
 <td>Repeat the eccentric contraction for 10 cycles with 2 min rest between cycles.</td>
 </tr>
 <tr>
 <td>6. CSA of the EDL muscle </td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>CSA = (muscle mass (g) / [1.06 g/cm3 x (Lo x 0.44)]. 1.06 g/cm3 is the muscle density and 0.44 is the EDL muscle fiber length to Lo ratio. </td>
 </tr>
 <tr>
 <td colspan="9">1.3.2 Measuring the passive properties of the EDL muscle </td>
 </tr>
 <tr>
 <td>1. Six-step stretching protocol</td>
 <td>Adjust resting tension to Lo</td>
 <td></td>
 <td></td>
 <td></td>
 <td>10% Lo</td>
 <td></td>
 <td>2 cm/sec</td>
 <td>Repeat the stretching protocol with an increment of 10% Lo till 160% Lo is reached. Alow 1.5 sec between stretch cycles.</td>
 </tr>
 <tr>
 <td>2. SRR</td>
 <td>Adjust resting tension to Lo</td>
 <td></td>
 <td></td>
 <td></td>
 <td>10% Lo</td>
 <td></td>
 <td>2 cm/sec</td>
 <td>SSR is calculated by dividing the difference in the stress with the time elapsed between two time points in a time frame. </td>
 </tr>
 <tr>
 <td colspan="9">2.3 Measuring the contractile properties of the TA muscle </td>
 </tr>
 <tr>
 <td>1. Warm up</td>
 <td>4.0</td>
 <td>150</td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Rest the muscle for 60 sec between each stimulus. </td>
 </tr>
 <tr>
 <td>2. Optimal muscle length (Lo)</td>
 <td>3.0, 4.0, 5.0, 6.0 and 7.0</td>
 <td>1</td>
 <td>0.2</td>
 <td>300</td>
 <td></td>
 <td></td>
 <td></td>
 <td>Allow the muscle to relax for 30 sec between each stimulus. Measure the muscle optimal length using a digital caliper.</td>
 </tr>
 <tr>
 <td>3. CSA of the TA muscle </td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 <td>CSA = (muscle mass (g) / [1.06 g/cm3 x (Lo x 0.6)]. 0.6 is the TA muscle fiber length to Lo ratio. </td>
 </tr>
 </tbody>
</table>
Table 2. Parameters for the evaluation of the mechanical properties of the EDL and TA muscles.
<table border="1">
 <tbody>
 <tr>
 <td>Strain </td>
 <td>Age (month) </td>
 <td>Body weight (g)</td>
 <td>EDL weight (mg)</td>
 <td>EDL Lo (mm)</td>
 <td>EDL CSA (mm2)</td>
 </tr>
 <tr>
 <td>BL10</td>
 <td>6</td>
 <td>32.03 &plusmn; 0.57</td>
 <td>13.90 &plusmn; 0.77</td>
 <td>14.09 &plusmn; 0.04</td>
 <td>2.12 &plusmn; 0.12</td>
 </tr>
 <tr>
 <td>mdx</td>
 <td>6</td>
 <td>35.44 &plusmn; 0.42*</td>
 <td>16.73 &plusmn; 0.42*</td>
 <td>13.93 &plusmn; 0.05*</td>
 <td>2.57 &plusmn; 0.07*</td>
 </tr>
 </tbody>
</table>
Table 3. Morphometric properties of the EDL muscle. *, the value in mdx mice is significantly different from that of age-matched BL10 mice.
<img src="/files/ftp_upload/50183/50183fig1.jpg" alt="Figure 1" fo:src="/files/ftp_upload/50183/50183fig1highres.jpg" fo:content-width="5.5in"/> 
 Figure 1. A schematic diagram of the custom-made mouse dissection board. The dissection board is made from a &frac12; inch thick plexiglass and was fabricated at the institutional shop. <a href="https://www.jove.com/files/ftp_upload/50183/50183fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50183/50183fig2.jpg" alt="Figure 2" fo:src="/files/ftp_upload/50183/50183fig2highres.jpg" fo:content-width="4in"/>/> 
 Figure 2. A series of digital images showing the steps of tying a double square knot followed by a loop knot at the MTJ. Asterisk, the EDL muscle; Arrow, the distal tendon of the EDL muscle.
<img src="/files/ftp_upload/50183/50183fig3.jpg" alt="Figure 3" fo:src="/files/ftp_upload/50183/50183fig3highres.jpg" fo:content-width="6in"/>/> 
 Figure 3. A schematic diagram of the custom-made platform for in situ TA muscle function assay. The plexiglass animal platform and the stainless steel knee holder were designed to mount on the 809B in situ mouse apparatus. *, Stainless steel rod (Cat# MPR-2.0, Siskiyou, Grants Pass, OR); #, Universal electrode holder (Cat# MXB, Siskiyou, Grants Pass, OR); &sect;, electrode attachment rod (Cat# MPR-3.0, Siskiyou, Grants Pass, OR); **, Sylgard block. <a href="https://www.jove.com/files/ftp_upload/50183/50183fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50183/50183fig4.jpg" alt="Figure 4" fo:src="/files/ftp_upload/50183/50183fig4highres.jpg" fo:content-width="4in"/>/> 
 Figure 4. Representative results for the contractile and passive properties of the EDL muscle. The contractile properties of the EDL muscle are characterized by the specific twitch force (A), the specific tetanic force (B), the time to peak tension (C) and the half relaxation time (D). The passive properties of the EDL muscle are assessed by the stress strain profile (E) and the SSR. *, mdx mice are significantly different from age-matched BL10 mice.

Watch this Scientific Journal Video about Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice at JoVE.com

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.

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 ...

evaluation-muscle-function-extensor-digitorum-longus-muscle-ex-vivo

Research

JoVE Journal

Biology

42.1K Views.  University of Missouri. 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.

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Medicine

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Bioengineering

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

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

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

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

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

Neuroscience

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

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. ...

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.

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 ...

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 ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

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 ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Myo-mechanical Analysis of Isolated Skeletal Muscle

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

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

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

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

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

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

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

In vivo Measurement of Knee Extensor Muscle Function in Mice