Name: human vastus lateralis skeletal muscle biopsy using the weil-blakesley conchotome
Uploaded: 2016-03-04T13:00:00
Description: Watch this Scientific Journal Video about Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome at JoVE.com

We wish to thank the study participants for making this work possible and the staff at the NIHR Southampton Biomedical Research Centre and the Wellcome Trust Clinical Research Facility, University Hospital Southampton for assistance with study measurements.
This work was supported by the NIHR Southampton Biomedical Research Centre, Nutrition, the MRC Lifecourse Epidemiology Unit and the University of Southampton. This report is independent research by the NIHR BRC Funding Scheme. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. AMB is supported by a NIHR Clinical Research Fellowship, HPP is supported by the NIHR Southampton Biomedical Research Centre.

Ethics statement: A description of the muscle biopsy procedure we follow within our institution now follows. The muscle biopsy procedure is used in the Hertfordshire Sarcopenia Study which has been approved by the Hertfordshire Research Committee number 07/Q0204/68. All participants gave written informed consent 20. 
NOTE: A single operator can perform the procedure, aided by an assistant who can engage the participant in conversation in order to reduce anxiety and ensure that they are comfortable at all ...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+needle-biopsy+of+skeletal+muscle+in+diagnosis+and+research.">Percutaneous needle-biopsy of skeletal muscle in diagnosis and research.</a> Lancet. 2 (7724), 593-595 (1971).</li><li>Edwards, R. H., Round, J. M., Jones, 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=Needle+biopsy+of+skeletal+muscle:+a+review+of+10+years+experience.">Needle biopsy of skeletal muscle: a review of 10 years experience.</a> Muscle Nerve. 6 (9), 676-683 (1983).</li><li>Shanely, R. A., Zwetsloot, K. A., Triplett, N. T., Meaney, M. P., Farris, G. E., Nieman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+muscle+biopsy+procedures+using+the+modified+Bergstrom+technique.">Human skeletal muscle biopsy procedures using the modified Bergstrom technique.</a> J Vis Exp. (91), e51812 (2014).</li><li>Coggan, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+biopsy+as+a+tool+in+the+study+of+aging.">Muscle biopsy as a tool in the study of aging.</a> J Gerontol A Biol Sci Med Sci. 50, 30-34 (1995).</li><li>Henriksson, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Semi-open'+muscle+biopsy+technique.+A+simple+outpatient+procedure.">'Semi-open' muscle biopsy technique. A simple outpatient procedure.</a> Acta Neurol Scand. 59 (6), 317-323 (1979).</li><li>Dorph, C., Nennesmo, I., Lundberg, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+conchotome+muscle+biopsy.+A+useful+diagnostic+and+assessment+tool.">Percutaneous conchotome muscle biopsy. A useful diagnostic and assessment tool.</a> J Rheumatol. 28 (7), 1591-1599 (2001).</li><li>Poulsen, M. B., Bojsen-Moller, M., Jakobsen, J., Andersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+conchotome+biopsy+of+the+deltoid+and+quadricep+muscles+in+the+diagnosis+of+neuromuscular+disorders.">Percutaneous conchotome biopsy of the deltoid and quadricep muscles in the diagnosis of neuromuscular disorders.</a> J Clin Neuromuscul Dis. 7 (1), 36-41 (2005).</li><li>Dietrichson, P., Mellgren, S. I., Skre, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+biopsy+with+the+percutaneous+conchotome+technique.">Muscle biopsy with the percutaneous conchotome technique.</a> J Oslo City Hosp. 30 (5), 73-79 (1980).</li><li>Dietrichson, P., Coakley, J., Smith, P. E., Griffiths, R. D., Helliwell, T. R., Edwards, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conchotome+and+needle+percutaneous+biopsy+of+skeletal+muscle.">Conchotome and needle percutaneous biopsy of skeletal muscle.</a> J Neurol Neurosurg Psychiatry. 50 (11), 1461-1467 (1987).</li><li>Andonopoulos, A. P., Papadimitriou, C., Melachrinou, M., Meimaris, N., Vlahanastasi, C., Bounas, A., Georgiou, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymptomatic+gastrocnemius+muscle+biopsy:+an+extremely+sensitive+and+specific+test+in+the+pathologic+confirmation+of+sarcoidosis+presenting+with+hilar+adenopathy.">Asymptomatic gastrocnemius muscle biopsy: an extremely sensitive and specific test in the pathologic confirmation of sarcoidosis presenting with hilar adenopathy.</a> Clin Exp Rheumatol. 19 (5), 569-572 (2001).</li><li>Helliwell, T. R., Coakley, J., Smith, P. E., Edwards, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+and+morphometry+of+the+normal+human+tibialis+anterior+muscle.">The morphology and morphometry of the normal human tibialis anterior muscle.</a> Neuropathol Appl Neurobiol. 13 (4), 297-307 (1987).</li><li>Patel, H. P., Jameson, K. A., Syddall, H. E., Martin, H. J., Stewart, C. E., Cooper, C., Sayer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+influences,+muscle+morphology,+and+sarcopenia+in+community-dwelling+older+men.">Developmental influences, muscle morphology, and sarcopenia in community-dwelling older men.</a> J Gerontol A Biol Sci Med Sci. 67 (1), 82-87 (2012).</li><li>Patel, H. P., Syddall, H. E., Martin, H. J., Stewart, C. E., Cooper, C., Sayer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hertfordshire+Sarcopenia+Study:+design+and+methods.">Hertfordshire Sarcopenia Study: design and methods.</a> BMC Geriatrics. 10 (1), 43 (2010).</li><li>Anderson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+biopsy+procedure+and+assessment+of+skeletal+muscle+biopsies.">Recommendations for the biopsy procedure and assessment of skeletal muscle biopsies.</a> Virchows Arch. 431 (4), 227-233 (1997).</li><li>Patel, H. P., Syddall, H. E., Martin, H. J., Cooper, C., Stewart, C., Sayer, A. 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+Feasibility+and+Acceptability+of+Muscle+Biopsy+in+Epidemiological+Studies:+Findings+from+the+Hertfordshire+Sarcopenia+Study+(HSS).">The Feasibility and Acceptability of Muscle Biopsy in Epidemiological Studies: Findings from the Hertfordshire Sarcopenia Study (HSS).</a> J Nutr Health Aging. 15 (1), 10-15 (2011).</li><li>Charifi, N., Kadi, F., Feasson, L., Costes, F., Geyssant, A., Denis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+microvessel+tortuosity+in+the+vastus+lateralis+muscle+of+old+men+in+response+to+endurance+training.">Enhancement of microvessel tortuosity in the vastus lateralis muscle of old men in response to endurance training.</a> J Physiol. 554 (Pt 2), 559-569 (2004).</li><li>Frontera, W. R., Suh, D., Krivickas, L. S., Hughes, V. A., Goldstein, R., Roubenoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber+quality+in+older+men+and+women.">Skeletal muscle fiber quality in older men and women.</a> Am J Physiol Cell Physiol. 279 (3), C611-C618 (2000).</li><li>He, J., Watkins, S., Kelley, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+lipid+content+and+oxidative+enzyme+activity+in+relation+to+muscle+fiber+type+in+type+2+diabetes+and+obesity.">Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity.</a> Diabetes. 50 (4), 817-823 (2001).</li><li>Patel, H. P., Al-Shanti, N., Davies, L. C., Barton, S. J., Grounds, M. D., Tellam, R. L., Stewart, C. E., Cooper, C., Sayer, A. 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To ensure participant safety and perform the muscle biopsy efficiently, it is vital to pay attention to critical steps within the protocol.
The participants must be selected after carefully considering the exclusion criteria to avoid complications such as bleeding and poor wound healing. Strict asepsis throughout the procedure is essential. Application of the correct technique will ensure participant comfort, minimize unwanted trauma to participant&#39;s tissues and allow a sufficient sample y...

Obtaining skeletal muscle for the diagnosis of a myopathy and other neuromuscular degenerative diseases relies on safe and efficient methods which are acceptable and not incapacitating to patients 1. Historically, methods of obtaining muscle tissue for analysis included open biopsies performed under general anesthesia or from post mortem. These techniques allowed direct visualization of the muscle and permitted a larger piece of muscle to be biopsied. Given the potential disadvantages these techniques have to the patient in terms of hospital stay and recovery, percutaneous &#39;semi-open&#39; muscle biopsy methods were introduced as alternatives. This technique was first performed by Duchenne (1806-1875), who used a self-constructed needle with a trocar and was able to obtain a muscle sample from a living subject without general anesthesia 2. Since then, various forms of biopsy needles have been used 3,4,5 . However, the biopsy needle developed by Bergstrom in 1962 has been the most commonly used in clinical practice; both in children and adults, as well as in research 6,7,8,9,10. It possesses a sharp trocar, a cutting cannula and a pushing rod to expel the tissue post biopsy. Muscle yields obtained from this procedure have been reported to range from 25 -293 mg after repeated sampling 11,9.
Henriksson introduced the Weil-Blakesley conchotome in 1979 as an alternative semi-open muscle biopsy method (Figure 1) 12. The instrument is a single structure unlike the Bergstrom construct and is designed like a forcep with a sharp biting tip. The tip size can range from 4 mm to 6 mm in width. When the two edges of the biting tip oppose, a hollow is formed that ensures capture of the muscle. The conchotome is inserted through a 5 - 10 mm skin incision and avoids the need for a trocar to penetrate the muscle and the overlying fascia as a scalpel can be used to make a track down to the muscle.
This method allows controlled tissue penetration with a high degree of maneuverability 13,14 . It is especially useful for sites where undue pressure could potentially damage neurovascular or underlying bony structures e.g., at the site of the tibialis anterior 15,14. Muscle yields using the conchotome can be variable and in our practice 20 - 200 mg of muscle has been obtained after repeated sampling. One potential advantage of the conchotome over the Bergstrom needle, in addition to the high degree of maneuverability it offers, is that the biting tip does not need to be sharpened or replaced as often as the trocar of the Bergstrom construct 16 .
The overall goal of muscle biopsy using the Weil Blakesley conchotome is to obtain sufficient amounts of muscle tissue to enable histochemistry and molecular analysis for both diagnostic and research study. It is a simple and safe procedure that can be learned easily. With this technique, muscle suitable for biopsy includes biceps, triceps, deltoid, gastrocnemius, tibialis anterior, soleus and the sacrospinals 17,15,7,18,14 . The outermost part of the vastus lateralis is the most common site used for biopsy as it avoids the main neurovascular structures within the thigh. It is identified approximately two thirds down an imaginary line joining the anterior superior iliac spine and the patella. Muscle morphology data, for example myofibre area, myofibre proportions, capillary density derived from vastus lateralis sampling are widely available in the literature allowing comparison between studies 11,19 .

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	<tbody>
		<tr>
			<td>Weil-Blakesley conchotome</td>
			<td>Gebr&#252;der Zepf Medizintechnik, D&#252;rbheim, Germany</td>
			<td>None</td>
			<td>6 mm biting tip</td>
		</tr>
		<tr>
			<td>5 ml 2% lidocaine</td>
			<td>Generic use</td>
			<td>None</td>
			<td>One 5ml vial should suffice for adequate anaesthesia</td>
		</tr>
		<tr>
			<td>Alcohol swab</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mls syringe</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>25G and 23G sterile needles</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel size 11</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze squares</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% 10 ml</td>
			<td>Fannin</td>
			<td>1122/002/001</td>
			<td></td>
		</tr>
		<tr>
			<td>Steri-Drape&#8482; Small Drape with Adhesive Aperture</td>
			<td>3M</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>ChloraPrep 2% w/v /70% v/v cutaneous solution&#160;</td>
			<td>CareFusion</td>
			<td>270400</td>
			<td>Two</td>
		</tr>
		<tr>
			<td>Steri-Strip&#8482; 1/4 inch x 3 in Reinforced Adhesive Skin Closures</td>
			<td>3M</td>
			<td>R1541</td>
			<td></td>
		</tr>
		<tr>
			<td>PRIMAPORE sterile dressing 15cm x 8cm</td>
			<td>Smith &#38;&#160; 
			Nephew</td>
			<td>66000318</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton crepe bandage Hospicrepe 233 7,5 cm x 4,5 m</td>
			<td>Hartmann</td>
			<td>915562</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible cohesive bandage Co-Plus LF</td>
			<td>BSN medical</td>
			<td>2904565</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable absorbent sheet</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore or transpore tape</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gown with sterile paper towels</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The procedure described above is safe and acceptable in both clinical and research settings. When the biopsy is performed as part of the diagnostic process, the muscle should be chosen according to patient&#39;s symptoms and signs of muscular weakness. The biopsy site should be free from previous injuries, contractures or instrumentation 21,1. In research, standardized conditions such as fasting or exclusion of patients with diabetes may be required 19. Anticoagulant...

Watch this Scientific Journal Video about Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome at JoVE.com

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.

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 ...

The overall goal of this method is to introduce a safe, effective, and well-tolerated technique of muscle biopsy of the vastus lateralis, to obtain sufficient tissue for histochemistry and molecular analysis in both diagnostic and research studies. This method can help investigators obtain sufficient muscle tissue with minimal discomfort and trauma for the participant. The main advantage of this technique is that it's safe and acceptable in both clinical and research settings.Complications are low, and discomfort's kept to a minimum when the procedure is performed using a standard technique. To begin, ask the participant to lie comfortably on a couch in a supine position. Expose the preferred thigh from the groin crease, and spread a disposable absorbent sheet under the exposed thigh.Ensure that the leg remains straight and relaxed throughout the procedure, but the thigh is tensed momentarily to outline the vastus lateralis. Mark the procedure site approximately 2/3 down an imaginary line from the anterior superior iliac spine to the patella. To prepare the skin, use a sterile shaving blade to shave approximately four centimeters in diameter surrounding the proposed biopsy site, in men, if required.Use an alcohol swab to clean the skin. Use a local anesthetic to infiltrate the skin and overlying fascia, then, with a 25-gauge needle, raise a subcutaneous bleb of 2%lidocaine. Next, use a 23-gauge needle to infiltrate deeper into the subcutaneous tissue, aiming to penetrate at least 1/2 an inch, depending on the subcutaneous content at the biopsy site.Allow two to five minutes for the anesthesia to take action, and confirm anesthesia by using a needle or scalpel blade tip to gently probe the skin. To prepare a sterile field, wash hands with soap and water, and maintain clean hands until a sterile gown is worn. Cover hands with sterile gloves before creating a sterile field on the procedure trolley.With 2%Chlorhexidine isopropyl alcohol solution, or an iodine-based skin disinfectant, sterilize the biopsy site. Apply a sterile drape with an adhesive aperture to expose the biopsy site and to maintain a sterile field. The first critical step is to puncture the skin and make a sufficiently deep track into and beyond the fascia.To perform the biopsy procedure, with a size 11 scalpel, make a five to 10 millimeter incision on the skin, and in through the overlying fascia. While holding and supporting the thigh surrounding the biopsy site, insert the closed biting tip of the conchotome through the incision, at a right angle to the long axis of the femur, approximately 1/2 to one inch deep. Next, rotate the conchotome from 90 to 180 degrees to cut the muscle, then withdraw the conchotome and open the tip onto sterile gauze dampened by sterile normal saline.Transfer the gauze into a container placed on ice. The second critical step is to insert the conchotome within the track, to maneuver the conchotome to ensure sufficient muscle has been obtained. If necessary, the procedure can be repeated within the track, and single wound site to obtain sufficient muscle tissue.Following the procedure, apply direct pressure to the wound for up to five minutes. Then place sterile strips parallel to the wound to close it. Place a sterile absorbent dressing on the Steri-Strips, and apply a two-layer bandage for compression, using tape to secure.Explain the wound dressing method to the participant for them to perform three to four days after the procedure. Instruct the participant to remove the compression bandage before going to bed later that evening. Explain that it is common to experience some thigh stiffness, which can be relieved by gentle exercise, such as walking.Explain that they can use acetaminophen for the pain. As shown in this table, in the Hertfordshire sarcopenia study, the pain score is measured using the pain Visual Analogue Scale of zero millimeters for no pain, to 100 millimeters to pain as bad as can be, were typically low, with a median of seven millimeters during the procedure, and four millimeters one day after the procedure. This suggests that the subjects in this research study found the procedure minimally painful.Shown here are representative tissue samples obtained through percutaneous muscle biopsy with the Weil-Blakesley conchotome. The samples obtained should resemble the muscle tissue in this panel. However, there is a high probability of obtaining subcutaneous fatty tissue, as seen in this panel.This figure shows a serial cross section with differential fiber staining. Darkly stained fibers represent type two fast fibers detected with an anti-myosin fast antibody. Once mastered, this technique can be done in 20 minutes, if it is performed properly.While attempting this procedure, it's important to remember to maintain a sterile field, and keep the participant engaged. Following this procedure, other methods like histochemistry and DNA or RNA extraction can be performed, and all that will answer additional questions, like what are the cellular and molecular changes that occur in aging muscle? After its development, this technique paved way for researchers in the field of muscle biology and aging, to explore the changes in muscle tissue with sarcopenia, the age-related decline of muscle mass and function.After watching this video, you should have a good understanding of how to perform a muscle biopsy of the vastus lateralis, using the Weil-Blakesley conchotome, a simple, effective, and well-tolerated procedure. Don't forget that working with sharp needles and instruments can be hazardous, and standard safety precautions as well as strict asepsis should always be maintained while performing this procedure.

human-vastus-lateralis-skeletal-muscle-biopsy-using-weil-blakesley

Research

JoVE Journal

Medicine

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 ...

In Vivo Canine Muscle Function Assay

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

In Vivo Canine Muscle Function Assay

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

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

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 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

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. ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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 ...

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 ...