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 times. The procedure room should be equipped with a sink, a height adjustable couch, clean surfaces and a procedure trolley.
1. Prepare the Participant (Figure 2)
<ol>
	<li>Ask the participant to lay comfortably, supine on a couch. Expose the preferred thigh from the groin crease. 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.</li>
	<li>Mark the procedure site approximately two-thirds down an imaginary line from the anterior superior iliac spine to the patella.</li>
</ol>
2. Prepare the Skin (Figure 2)
<ol>
	<li>Shave the skin to approximately 4 cm&#160;in diameter surrounding the proposed biopsy site with a sterile shaving blade and clean the skin with an alcohol swab. 
	NOTE: This is to prevent any subsequent hair entrapment in the wound during the healing process. This step is optional according to the operator&#39;s preference.</li>
	<li>Infiltrate the skin and overlying fascia with local anesthetic. Use a 25 G needle to raise a subcutaneous bleb of 2% lidocaine then infiltrate deeper into the subcutaneous tissue with a 23 G needle. Aim to penetrate to at least half to one inch depending on the subcutaneous tissue content at the biopsy site. This depth should be sufficient to ensure the overlying fascia is anesthetized. Allow 2 - 5 min&#160;for the anesthetic to action. Confirm anesthesia by probing the skin gently with the needle or scalpel blade tip.</li>
</ol>
3. Prepare a Sterile Field (Figure 2)
<ol>
	<li>Wash hands with soap and maintain clean hands until a sterile gown is worn. Cover hands with sterile gloves before creating a sterile field on the procedure trolley.</li>
	<li>Sterilize the biopsy site with 2% chlorhexidine/isopropyl alcohol solution or an iodine based skin disinfectant. Apply a sterile drape with an adhesive aperture to expose the biopsy site but to also maintain a sterile field.</li>
</ol>
4. Perform the Procedure (Figure 2)
<ol>
	<li>With a size 11 scalpel, make a 5 - 10 mm incision on the skin and down and through the overlying fascia. Insert the closed biting tip of the conchotome through the incision at a right angle to the long axis of the femur, to a depth averaging half to one inch. If required use the free hand to hold and support the thigh surrounding the biopsy site while the tip of the conchotome is opened and closed within the muscle.</li>
	<li>Rotate the conchotome through 90 - 180&#176; to cut the muscle. Withdraw the conchotome and open the tip onto sterile gauze dampened by sterile normal saline. Repeat the procedure within the single wound site, if necessary, to obtain sufficient muscle tissue. Transfer the gauze into a container placed on ice. Transfer the container to a preparation laboratory to process the muscle according to the local institutional protocol.</li>
</ol>
5. Post Procedure (Figure 2)
<ol>
	<li>Apply direct pressure to the wound for up to 5 min. Close the wound with steri-strips by placing them parallel to the wound as opposed to right angles to avoid potentially removing scar tissue and reopening the wound if the steri-strips are removed inadvertently. Place a sterile absorbent dressing on the steri-strips and tie a two-layer bandage for compression and secure with tape.</li>
	<li>Explain the wound dressing method to the participant as they will have to perform a dressing change 3 - 4 days after the procedure. Ensure that the participant removes the compression bandage before going to bed later that evening.</li>
</ol>
6. Post Biopsy Advice
<ol>
	<li>Inform the participant that it is common to experience some thigh stiffness which can be relieved by gentle exercise (e.g., walking). Use simple analgesia such as acetaminophen. Warn the participant about transient numbness around the biopsy site that may persist for up to two weeks as well as the potential complication of wound infection.</li>
</ol>
7. Instructions for the Subject Post Procedure
<ol>
	<li>Ask the participant to avoid vigorous activity for 72 hr (such as climbing, running, heavy lifting). Ask the participant to avoid immersion in water for 48 - 72 hr. However, instruct the participant to wrap cling film or similar around the biopsy site when having a shower to keep the dressing dry.</li>
	<li>Ask the participant to change the dressing after 3 - 4 days and remove the steri-strips after one week. 
	NOTE: The participant should be given written instructions for post biopsy care, spare dressings and emergency contact details of the team responsible for performing the procedure.</li>
</ol>

<ol>
	<li>Dubowitz, V., Sewry, C. A., Oldfords, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Muscle Biopsy: A Practical Approach. , (2013).</li><li>Parent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duchenne+De+Boulogne:+a+pioneer+in+neurology+and+medical+photography.">Duchenne De Boulogne: a pioneer in neurology and medical photography.</a> Can J Neurol Sci. 32 (3), 369-377 (2005).</li><li>Hayot, M., Michaud, A., Koechlin, C., Caron, M. A., Leblanc, P., Prefaut, C., Maltais, F. <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+microbiopsy:+a+validation+study+of+a+minimally+invasive+technique.">Skeletal muscle microbiopsy: a validation study of a minimally invasive technique.</a> Eur Respir J. 25 (3), 431-440 (2005).</li><li>O'Rourke, K. S., Blaivas, M., Ike, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+needle+muscle+biopsy+in+a+university+rheumatology+practice.">Utility of needle muscle biopsy in a university rheumatology practice.</a> J Rheumatol. 21 (3), 413-424 (1994).</li><li>O'Rourke, K. S., Ike, R. W. <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.">Muscle biopsy.</a> Curr Opin Rheumatol. 7 (6), 462-468 (1995).</li><li>Bergstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+electrolytes+in+man.">Muscle electrolytes in man.</a> Scand J Clin Lab Invest. 14, (1962).</li><li>Edwards, R., Young, A., Wiles, M. <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+in+the+diagnosis+of+myopathy+and+the+clinical+study+of+muscle+function+and+repair.">Needle biopsy of skeletal muscle in the diagnosis of myopathy and the clinical study of muscle function and repair.</a> N Engl J Med. 302 (5), 261-271 (1980).</li><li>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=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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lean+mass,+muscle+strength+and+gene+expression+in+community+dwelling+older+men:+findings+from+the+Hertfordshire+Sarcopenia+Study+(HSS).">Lean mass, muscle strength and gene expression in community dwelling older men: findings from the Hertfordshire Sarcopenia Study (HSS).</a> Calcif Tissue Int. 95 (4), 308-316 (2014).</li><li>Peterson, C. M., Johannsen, D. L., Ravussin, 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+mitochondria+and+aging:+a+review.">Skeletal muscle mitochondria and aging: a review.</a> J Aging Res. 2012, 194821 (2012).</li></ol>

None declared.

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 .

<table><tbody><tr><td>Weil-Blakesley conchotome</td><td>Gebr&amp;uuml;der Zepf Medizintechnik, D&amp;uuml;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 5 ml 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 ml syringe</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>25 G and 23 G 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&amp;trade; 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&amp;nbsp;</td><td>CareFusion</td><td>270400</td><td>Two</td></tr><tr><td>Steri-Strip&amp;trade; 1/4 inch x 3 inch Reinforced Adhesive Skin Closures</td><td>3M</td><td>R1541</td><td></td></tr><tr><td>PRIMAPORE sterile dressing 15 cm x 8 cm</td><td>Smith &amp;amp;&amp;nbsp;&lt;br/&gt; 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'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. Anticoagulants su...

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 ...

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

Research

JoVE Journal

Medicine

16.7K Views.  University of Southampton, University Hospital Southampton. This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

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. ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

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.

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 ...

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 ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Biology

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

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

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