This work was supported by a Public Health Service Grant (HL086513) from NHLBI to P.E.O, and a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to H.S.B.
S.C. was supported by a California Institute for Regenerative Medicine Bridges to Stem Cell Research Award (TB1-01194) to San Francisco State University.

The protocol is performed with the approval of the UCSF Institutional Animal Care and Use Committee (IACUC).
1. Dissection of the Mouse Extensor Digitorum Longus (EDL) Muscle
<ol>
	<li>Perform all animal procedures in accordance with institutional guidelines.</li>
	<li>Euthanize animal with 200 mg/kg intraperitoneal pentobarbital/cervical dislocation just prior to muscle harvest 10. The dissection should be well practiced so that the muscle can be harvested and mounted in the tension transducer within 15 minutes of euthanasia.</li>
	<li>Arrange carcass supine on dissection tray and pin leg to tray.</li>
	<li>Under dissection microscope, open skin, carefully open fascia (Fig.1A), and peel tibialis from the ankle upwards to expose EDL (Fig.1B). Use drops of lactated Ringer's solution to keep the muscle moist and buffered during the harvest.</li>
	<li>Remove EDL, preserving as much tendon as possible on each end, and put into a Petri dish containing lactated Ringer's solution. Tie suture to each of the muscle tendons (Fig.1C). It is essential that the muscle fibers not be touched or disturbed during the dissection.</li>
</ol>
2. Mounting of the Mouse EDL in the Muscle Strip Myograph
<ol>
	<li>For these studies, a tissue bath is needed that secures the muscle while allowing it to bathe in physiological solution at constant temperature with continuous oxygenation. The bath is coupled with a force transducer for the measurement of muscle tension. We employ an integrated muscle strip myograph bath from Danish Myo Technology (DMT Model 820MS) for this purpose. In addition, a square pulse electrical stimulator (Grass Model S48) and data acquisition platform (ADInstruments PowerLab Data Acquisition System and LabChart software) are needed to elicit, record and analyze myo-mechanical responses, respectively. The DMT 820MS has platinum electrodes integrated into the chamber cover that are positioned on either side of the muscle, in the mid-portion of the muscle strip. Other myographs may require specific attention to electrode placement.</li>
	<li>Fill myograph bath with 5 mL Krebs Henseleit solution 11. Warm to 25&deg;C. Bubble O2/CO2 (95%/5%) through bath for 15 minutes prior to use.</li>
	<li>Use sutures to extend EDL between clamps of myograph and secure the tendons of the EDL muscle (Fig.1D,E) between the clamps. Be careful not to clamp the muscle itself.</li>
	<li>Maintain myograph bath at 25&deg;C.</li>
</ol>
3. Myo-mechanical Analysis
A. Twitch tension
<ol>
	<li>Set initial length in the bath so that there is no muscle laxity.</li>
	<li>Determine maximal stimulus (duration 0.5 ms) by adjusting voltage in order to obtain maximal twitch tension, then set stimulus at 20% above maximal (to achieve supramaximal stimulus). In our studies, the supramaximal stimulus is usually achieved at an output of 40 volts.</li>
	<li>Verify output from the stimulator using an oscilloscope.</li>
	<li>Determine optimal length by gradually stretching the muscle until there is no further increase in twitch tension.</li>
	<li>Allow muscle to equilibrate for 3 min.</li>
	<li>Deliver supramaximal square stimulus (0.5 ms) at optimal length using Grass S44 electronic stimulator, and record output.</li>
	<li>Record: twitch tension curve (Pt vs time; Fig.2A).</li>
</ol>
B. Tetanus tension
<ol>
	<li>Allow muscle to rest for 3 minutes.</li>
	<li>Apply a train of supramaximal stimuli for 300msec at 150 Hz at optimal length using Grass S44 electronic stimulator, and record output.</li>
	<li>Record: tetanus tension curve (Po vs time; Fig.2B).</li>
</ol>
C. Force-frequency
<ol>
	<li>Allow muscle to rest for 3 minutes.</li>
	<li>Force-frequency: apply trains of supramaximal stimuli at 30, 60, 100, 140, and 160 Hz with 3 minutes rest between each stimulus (Fig.3). Alternatively, trains may be applied 15, 25, 35, 45, 55, 65, 75, 100, 140 and 160 Hz for better resolution at lower frequencies, where force changes substantially.</li>
	<li>Plot: force-frequency relationship (% maximal force vs stimulation frequency).</li>
</ol>
D. Fatigue
<ol>
	<li>Apply train of short tetani: 60 Hz for 300 ms (or frequency adjusted to produce 50% of peak force), every 3 seconds for 10 minutes. By 10 minutes, the tetanic force should decline to a plateau level of ~15% of the initial value (Fig.4).</li>
	<li>Plot: low frequency fatigue (% maximal force vs time).</li>
</ol>
E. Additional data collection at end of protocol
<ol>
	<li>Before unmounting the muscle from the myograph, set the muscle at optimal length as determined in step III.A.4 and measure its diameter using either an ocular on the microscope or with calipers. Calculate cross-sectional area (&mu;m2).</li>
	<li>Measure muscle mass (mg) by removing sutures and weighing muscle.</li>
	<li>Weigh mouse to assess body mass (gm).</li>
</ol>
4. Calculations
<ol>
	<li>Muscle:body mass ratio = 
	muscle mass/body mass</li>
	<li>Twitch tension, Pt (mN) = 
	maximum tension generated during twitch</li>
	<li>Specific twitch tension (N/cm2) = 
	twitch tension (mN)/cross-sectional area (&mu;m2) x 105 N/mN&bull;&mu;m2/cm2</li>
	<li>Time to peak tension (ms) = 
	time from onset of contraction to maximum tension</li>
	<li>Half-relaxation time (ms) = 
	time from peak tension to 50% of peak tension</li>
	<li>Tetanic tension, PO (mN) = 
	maximum tension generated during tetanus</li>
	<li>Specific tetanic tension (N/cm2) = 
	tetanic tension (mN)/cross-sectional area (&mu;m2) x 105 N/mN&bull;&mu;m2/cm2</li>
	<li>Maximal rate of rise of tetanus (N/s) = 
	maximum rate of increase in tension during the tension rise in tetanus, i.e., the maximum slope of the tetanic tension curve (or, dPO/dt)</li>
	<li>Half-relaxation tetanic tension (ms) = 
	time from cessation of stimulation to 50% of the tension at termination of stimulation</li>
	<li>Twitch tension-to-tetanic tension ratio, Pt/PO = 
	maximum twitch tension/maximum tetanic tension</li>
	<li>Fatigue index= 
	ratio of tension after two minutes of low frequency fatigue to maximal isometric tension</li>
</ol>
5. Representative Results
<img src="/files/ftp_upload/2582/2582fig1.jpg" alt="Figure 1" /> 
Figure 1. Dissection of EDL muscle. A, Exposure of hindlimb muscles.TA, tibialis anterior. B, Exposure of EDL (extensor digitorum longus) muscle.C, Attachment of sutures to EDL tendons.D, Tension transducer bath (view from side). E, EDL mounted in bath (view from above). The muscle in incompletely immersed in the buffer for illustrative purposes; in practice, the muscle should be completely immersed to prevent from drying out.
<img src="/files/ftp_upload/2582/2582fig2.jpg" alt="Figure 2" /> 
Figure 2. Example of tension curves. A, Example of twitch tension curve illustrating maximal twitch tension (Pt), contraction time (CT) and half-relaxation time (HRT).Bar, 1s. B, Example of tetanic tension curve showing maximal tetanic tension (Po) and half-relaxation tetanic tension (HRTT). Bar, 1s.
<img src="/files/ftp_upload/2582/2582fig3.jpg" alt="Figure 3" /> 
Figure 3. Example of force-frequency relationship analysis. A, Tensions generated by incremental stimulation frequencies.B, Example of pulse train at 30MHz. Bar, 80ms. C, Example of pulse train at 140MHz. Bar, 80ms. D, Example of force-frequency curve derived from data shown in A. The shape of the force-frequency curve is characteristic of muscle strength, and can be compared between muscles from different animals.
<img src="/files/ftp_upload/2582/2582fig4.jpg" alt="Figure 4" /> 
Figure 4. Example of low frequency fatigue analysis. A, Decremental tensions generated over period of low frequency stimulation.Examples of pulse trains at indicated time points (B,C,D) are shown below. E, Example of low frequency fatigue curve derived from data shown in A. The shape of the low frequency fatigue curve is characteristic of muscle strength, and can be compared between muscles from different animals.

<ol>
	<li>Harcourt, L. J., Schertzer, J. D., Ryall, J. G., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+dose+formoterol+administration+improves+muscle+function+in+dystrophic+mdx+mice+without+increasing+fatigue.">Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue.</a> Neuromuscul Disord. 17, 47-55 (2007).</li><li>Messina, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+overexpression+via+adeno-associated+virus+gene+transfer+promotes+skeletal+muscle+regeneration+and+enhances+muscle+function+in+mdx+mice.">VEGF overexpression via adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle function in mdx mice.</a> FASEB J. 21, 3737-3746 (2007).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+1-phosphate+protects+mouse+extensor+digitorum+longus+skeletal+muscle+during+fatigue.">Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue.</a> Am J Physiol Cell Physiol. 288, C1367-C1373 (2005).</li><li>MacIntosh, B. R., Willis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-frequency+relationship+and+potentiation+in+mammalian+skeletal+muscle.">Force-frequency relationship and potentiation in mammalian skeletal muscle.</a> J Appl Physiol. 88, 2088-2096 (2000).</li><li>Hayes, A., Williams, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+clenbuterol-treated+mdx+muscle+are+enhanced+by+low-intensity+swimming.">Contractile properties of clenbuterol-treated mdx muscle are enhanced by low-intensity swimming.</a> J Appl Physiol. 82, 435-439 (1997).</li><li>Coulton, G. R., Curtin, N. A., Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+II.+Contractile+properties.">The mdx mouse skeletal muscle myopathy: II. Contractile properties.</a> Neuropathol Appl Neurobiol. 14, 299-314 (1988).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+alpha-sarcoglycan+differently+affects+fast-+and+slow-twitch+skeletal+muscles.">Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles.</a> Am J Physiol Regul Integr Comp Physiol. 289, R1328-R1337 (2005).</li><li>Chan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+for+speed:+contractile+properties+of+isolated+whole+EDL+muscle+from+an+alpha-actinin-3+knockout+mouse.">A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse.</a> Am J Physiol Cell Physiol. 295, C897-C904 (2008).</li><li>Personius, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grip+force,+EDL+contractile+properties,+and+voluntary+wheel+running+after+postdevelopmental+myostatin+depletion+in+mice.">Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice.</a> J Appl. , (2010).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Curr Protoc Immunol. Chap 1, (2006).</li><li>Beekley, M. D., Wetzel, P., Kubis, P., Gros, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscle+fibre+bundles+from+mice+deficient+in+carbonic+anhydrase+II.">Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II.</a> Pflugers Arch. 452, 453-463 (2006).</li></ol>

No conflicts of interest declared.

We describe a detailed approach to evaluating myo-mechanical properties in explanted hindlimb muscle from the mouse. The EDL, while more difficult to dissect because of its posterior position behind the tibialis anterior muscle, is easier to evaluate than the tibialis anterior because of its prominent tendinous attachments to the ankle and knee joints. These tendons facilitate mounting in the muscle strip myograph. In contrast, the more easily accessed tibialis anterior has a broad, almost atendinous attachment at the knee joint, making it exceptionally difficult to both dissect without compromising the muscle, and mount securely in the myograph. We also point out that rapidly mounting the muscle in an oxygenated bath in physiological buffer and temperature is essential to preserving the muscle's mechanical properties. We have found that we can repeat this analysis for up to 30 minutes without significant changes in muscle response under these conditions. Finally, it is essential that the muscle fibers not be touched during the dissection and mounting procedures, as this can have adverse effects on muscle function, and result in underestimation of myo-mechanical force. By following these procedures, this analysis provides a robust quantitative approach to evaluating the effects of genetic modification on muscle function 6,7,8,9, as well as comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5.

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>	
<tr>
<td>5-0 silk sutures</td>
<td>&nbsp;</td>
<td>Oasis</td>
<td>MV682</td>
<td>General surgery</td>
</tr>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>General surgery</td>
</tr>
<tr>
<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
</tr>
<tr>
<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Lab Chart software</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Version 7</a></td>
<td>Data analysis</td>
</tr>
<tr>
<td>Muscle Strip Myograph</td>
<td>&nbsp;</td>
<td>DMT</td>
<td>820MS</td>
<td>Tension transduction</td>
</tr>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
</tr>
<tr>
<td>Oscilloscope</td>
<td>&nbsp;</td>
<td>EZ</td>
<td>OS-5020</td>
<td>Tension stimulation</td>
</tr>
<tr>
<td>Pentobarbital, sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3761</td>
<td>Euthanasia</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/PL3508/" target="_blank">PowerLab </a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a></td>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/PL3508/" target="_blank">8/30</a></td>
<td>Data acquisition </td>
</tr>
<tr>
<td>Square Pulse Stimulator</td>
<td>&nbsp;</td>
<td>Grass Tech.</td>
<td>S48</td>
<td>Tension stimulation</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>General surgery</td>
</tr>		</tbody>
		</table>
			Solutions and Media
Lactated Ringer's solution
<ul>
<li>100 mM NaCl</li>
<li>30 mM CH3CH(OH)COONa (sodium lactate)</li>
<li>4 mM KCl</li>
<li>1 mM CaCl2 2H2O (calcium chloride dihydrate)
<ul>
<li>adjust pH to 6.75</li>
</ul>
</li>
</ul>
Krebs Henseleit solution
<ul>
<li>118 mM NaCl</li>
<li>4.7 mM KCl</li>
<li>1.25 mM CaCl2</li>
<li>1.2 mM MgCl2</li>
<li>1.2 mM KH2PO4</li>
<li>25 mM NaHCO3</li>
<li>11 mM glucose
<ul>
<li>adjust pH to 7.2-7.4 by equilibrating with O2/CO2 (95%/5%) gas</li>
</ul>
</li>
</ul>
Pentobarbital
<ul>
<li>5 mg/ mL working solution in sterile water</li>
</ul>

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.

Watch this Scientific Journal Video about Myo-mechanical Analysis of Isolated Skeletal Muscle at JoVE.com

Myo-mechanical Analysis of Isolated Skeletal Muscle

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 ...

myo-mechanical-analysis-of-isolated-skeletal-muscle

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JoVE Journal

Medicine

26.7K 조회수.  University of California San Francisco. 을 평가하기 위해 생체내에 효과, 치료 방법은 근육의 힘 생성 및 fatigability을 quantitate 필요합니다. 우리는 세부 explanted 마우스 hindlimb 근육의 묘 - 기계적 성질을 평가하는 방법을. 이 분석은 유전자 근육 기능에 수정뿐만 아니라 근육 질환의 마우스 모델 요법의 비교의 효과를 quantitating하는 강력한 방법을 제공합니다.

비디오: Myo-mechanical Analysis of Isolated Skeletal Muscle

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

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 ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force generating capacity. However, over 90% of all upper limb muscles and 85% of all lower limb muscles have optimal fascicle lengths longer than the field-of-view of common traditional ultrasound (T-US) probes. A newer, less frequently adopted method called extended field-of-view ultrasound (EFOV-US) can enable direct measurement of fascicles longer than the field-of-view of a single T-US image. This method, which automatically fits together a sequence of T-US images from a dynamic scan, has been demonstrated to be valid and reliable for obtaining muscle fascicle lengths in vivo. Despite the numerous skeletal muscles with long fascicles and the validity of the EFOV-US method for making measurements of such fascicles, few published studies have utilized this method. In this study, we demonstrate both how to implement the EFOV-US method to obtain high quality musculoskeletal images and how to quantify fascicle lengths from those images. We expect that this demonstration will encourage the use of the EFOV-US method to increase the pool of muscles, both in healthy and impaired populations, for which we have in vivo muscle fascicle length data.

This study describes how to obtain high quality musculoskeletal images using the extended field-of-view ultrasound (EFOV-US) method for the purpose of making muscle fascicle length measures. We apply this method to muscles with fascicles that extend past the field-of-view of common traditional ultrasound (T-US) probes.