Name: human skeletal muscle biopsy procedures using the modified bergström technique
Uploaded: 2014-09-10T14:00:00.000+00:00
Duration: 7 min 20 s
Description: Watch this Scientific Journal Video about Human Skeletal Muscle Biopsy Procedures Using the Modified BergstrÃƒÂ¶m Technique at JoVE.com

No funding to declare.

The skeletal muscle procedure as described follows the guidelines of the Appalachian State University Institutional Review Board.
NOTE: The team consists of an operator (Ph.D. or M.D. trained in the biopsy technique), and at least one, but ideally, three or more technicians. The operator is responsible for conducting and overseeing all aspects of the skeletal muscle biopsy procedure. One technician (technician #1) is immediately responsible for assisting the operator with all aspects of muscle extraction. This includes being the &#8220;non-sterile hands&#8221; and applying suction with a sterile syringe. A second technician (technician #2) engages in conversation with subject and massages the subject&#8217;s opposite leg during procedure to minimize the subject&#39;s anxiety. The third technician (technician #3) is responsible for handling and processing the skeletal muscle sample. Downstream analytical measures dictate how many additional technicians are required for processing/preparing the skeletal muscle sample.
1. Subject Preparation
<ol>
	<li>Gather, prepare, and organize the materials specified for the muscle biopsy procedure (Table 1). Decontaminate and sterilize the Bergstr&#246;m needle (outer cannula and inner trocar) and the plunger according to standard practice16.</li>
	<li>Review the consent form with the subject. The skeletal muscle biopsy portion of the Consent to Participate form used at Appalachian State University is provided in online supplement 1.
	<ol>
		<li>Talk to the subject about the general aspects of the muscle biopsy procedure with a focus on possible risks of the procedure, skin preparation, application of the anesthetic, typical sensations. Instruct the subject to carefully read the Consent to Participate form and sign it to confirm that the study design and procedures are understood.</li>
	</ol>
	</li>
	<li>Confirm that the subject is not allergic to &#8220;___caine&#8221;-type medications (e.g., lidocaine).</li>
	<li>Instruct the subject to lie supine on a padded table with the thigh exposed. Place two disposable absorbent underpads with plastic backing under the subject&#8217;s leg. Position the leg in a relaxed manner with a small towel roll under the heel, such that the knee is fully extended (elevated ~1 cm), thus placing the vastus lateralis in a shortened position.</li>
	<li>Have the operator instruct the subject to momentarily contract the exposed thigh muscle so that the biopsy site can be visualized. Note: The vastus lateralis biopsy site is just anterior to the fascia lata (illiotibial band), approximately one-third of the distance between the top of the patella and the greater trochanter7.</li>
	<li>Have the operator mark just below (~0.5 cm) the incision site with a fine point permanent marker.</li>
	<li>Have the operator determine the approximate skinfold thickness by pinching the skinfold.</li>
	<li>Have the operator and technicians wash their hands with soap and warm water and don disposable gloves.</li>
	<li>Have the operator remove the hair from a region ~15 cm x 15 cm around the biopsy site (via clippers). Note: clipping the hair from the biopsy site prevents hair from getting into the incision during the procedure and during closing, and hair removal allows for better contact between the tape closures and the skin (see 3.4).</li>
	<li>Have the operator sterilize the area with swabs pre-soaked with a topical antiseptic, (such as povidone-iodine or chlorhexidine gluconate for subjects allergic to iodine/shellfish). Begin in the center and work in concentric circles towards the outer edge of the clipped area. At a minimum, repeat twice more with a new pre-soaked swab each time.</li>
</ol>
2. Biopsy Procedure
<ol>
	<li>Have the operator remove disposable gloves, wash hands with soap and warm water, and don sterile surgical gloves using aseptic technique.</li>
	<li>Have technician #1 present the sterile fenestrated drape to the operator. Have the operator, using aseptic technique, place the drape over the biopsy site to maintain a sterile field.</li>
	<li>Have technician #1 present the operator a 5 ml syringe fitted with a 21 G needle while maintaining aseptic technique. Clean the top of the lidocaine vial with an alcohol swab.
	<ol>
		<li>Have the operator immediately withdraw 5 ml of lidocaine. Remove the 21 G needle from the syringe and discard the needle into a sharps container.</li>
		<li>Have technician #1 present the operator with a 1-&#189; inch 25 G needle while maintaining aseptic technique. Have the operator place the 1-&#189; inch needle onto the syringe and evacuate the air bubbles from the syringe.</li>
	</ol>
	</li>
	<li>Have technician #1 spray ethyl chloride on the incision site (~0.5 cm above the indelible ink mark on the skin) until the skin appears to &#8220;blanch.&#8221;
	<ol>
		<li>Have the operator insert the needle approximately horizontal to the skin into the dermis, aspirate the needle, and then infiltrate with ~100 &#181;l of lidocaine to produce a &#8220;bleb&#8221; 2-4 mm in diameter.
		<ol>
			<li>Have the operator aspirate the needle (slightly withdraw the plunger of the syringe) to confirm that the needle has not been placed in a blood vessel. If blood appears in the syringe, withdraw the needle, discard in a sharps container, and begin again at 2.3.</li>
			<li>Have the operator advance the needle into the subcutaneous tissue, aspirate the needle, and then infiltrate the tissue with ~1 ml of lidocaine to form a bleb. Once the bleb has subsided, insert the 1-&#189; inch needle on the lidocaine-loaded syringe vertically into the incision site, stopping superficial to the fascia. Note: The subject may feel a slight momentary stinging sensation upon the initial injection of lidocaine (similar to a bee sting).</li>
		</ol>
		</li>
		<li>Ensure that the operator does not infiltrate the muscle with lidocoine because it is myotoxic17-19.</li>
		<li>Have the operator aspirate the needle and then slowly inject the remaining 4 ml of lidocaine while withdrawing the needle from the thigh. Place sterile gauze over injection site and allow the subject to relax while the local anesthetic takes effect.</li>
	</ol>
	</li>
	<li>After 2-3 min, have technician #1 present the operator a scalpel while maintaining aseptic technique.
	<ol>
		<li>Have the operator lightly probe the biopsy site with the tip of the scalpel to confirm that the area is anesthetized. If necessary, inject another 3-4 ml of lidocaine into the incision site, see 2.4.1.2.</li>
	</ol>
	</li>
	<li>Have the operator make a straight 1-cm incision through the skin and subcutaneous tissues (~2-3 mm above the ink mark) parallel to the femur.
	<ol>
		<li>Have the operator insert the scalpel deeper to make an incision through the fascia into the muscle. Do this once in each direction. Note: The subject may feel a twinge or pressure with the deeper cut if the scalpel blade cuts into the muscle.</li>
		<li>Have technician #2 engage the subject in conversation and massage the opposite leg.</li>
	</ol>
	</li>
	<li>Have the operator place a liberal amount of sterile gauze over the incision and biopsy site and apply direct pressure to the incision to reduce bleeding.
	<ol>
		<li>Have technician #1 connect the 3-way metal stopcock to the disposable 60 ml syringe and the 30 cm extension tubing (Figure 1).</li>
		<li>Have technician #1 connect one end of the tapered plastic tubing connecter into the free end of the extension tubing and firmly insert the other end of the tapered plastic tubing connecter into the large end of a 200 &#181;l pipette tip with ~15-18 mm of the tip cut off.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51812/51812fig1highres.jpg" width="500" /> 
Figure 1. The Bergstr&#246;m needle. (A) The Bergstr&#246;m needle (5 mm) is composed of the (a) outer cannula and (b) inner trocar, the associated components include (c) plunger, (d) 200 &#181;l pipette tip with ~15-18 mm cut off, (e) tapered plastic connector, (f) 3-way metal stopcock, (g) 30 cm extension tubing, and (h) disposable 60 ml syringe. (B) The Bergstr&#246;m needle (5 mm) and associated components assembled. (C) The inner trocar of the Bergstr&#246;m needle (5 mm) withdrawn approximately 1 cm opens the window of the outer cannula.
<ol start="9">
	<li>After the bleeding has subsided, have technician #1 open the sterilized autoclave pouch and present the 5 mm biopsy needle and inner trocar (Figure 1) to the operator while maintaining aseptic technique.
	<ol>
		<li>Have the operator assemble the needle (outer cannula and inner trocar), check alignment and sliding action.</li>
	</ol>
	</li>
	<li>Have the operator firmly hold the needle assembly (outer cannula and inner trocar) with both hands to prevent the inner trocar from rotating. Have technician #1 firmly insert the cut pipette tip into the top of the inner trocar portion of the biopsy needle with a slight downward twisting motion. Ensure that technician #1 does not touch the operator or the biopsy needle, only the pipette tip.</li>
	<li>Have the operator introduce the biopsy needle into the tissue through the incision. Locate the incision in the fascia (an acquired &#8220;feel&#8221;) with the tip of the biopsy needle. Advance the needle just past the fascia and then angle the needle downward toward the floor as the needle is advanced into the muscle. Note: Subjects may experience some mild discomfort (e.g., &#8220;deep pressure,&#8221; or mild cramping sensation) as the incision through the fascia is located with the tip of the biopsy needle and the needle is advanced into the muscle.
	<ol>
		<li>Have technician #2 instruct the subject to stay &#8220;relaxed&#8221; and not to contract their thigh muscles while the operator is advancing the needle into the muscle.</li>
	</ol>
	</li>
	<li>Once the biopsy needle is in position, have the operator signal technician #1 to open the disposable 60 ml syringe.
	<ol>
		<li>Have technician #1 rapidly pull the disposable 60 ml syringe open to the 40-50 cc mark to create suction within the biopsy needle.</li>
		<li>Have the operator pull the inner trocar out approximately 1 cm to open the window of the outer cannula while maintaining the position of the outer trocar within the muscle. Have the operator rapidly close the inner trocar to cut (clip) and collect the muscle sample. Have the operator signal technician #1 to release suction.</li>
		<li>Have technician #1 open the stopcock to the room to release the suction.</li>
		<li>Have the operator rotate the biopsy needle 90&#176; and repeat the process. If required, have the operator repeat the rotation procedure up to 3x (for a total of 4 clips).</li>
	</ol>
	</li>
	<li>Have the operator apply direct counter-pressure to the incision site with sterile gauze while removing the biopsy needle from the thigh, being careful not to catch fascia or skin. If significant resistance is felt, re-cut while applying a twisting motion to the inner trocar.</li>
	<li>Upon removal of the biopsy needle from the thigh, have the operator pull the inner trocar back 1 cm to visually inspect the lumen of the outer cannula to estimate if an adequate amount of tissue was collected.
	<ol>
		<li>Have the operator hand the biopsy needle to technician #1. To maintain aseptic technique ensure that technician #1 only touches the needle, not the operator. Have technician #1 hand the biopsy needle to technician #3.</li>
	</ol>
	</li>
	<li>Have technician #3 remove the tissue from the biopsy needle.
	<ol>
		<li>Have technician #3 use the plunger (Figure 1) and a pair of fine tip forceps to ensure that all muscle tissue is removed from the inner trocar and outer cannula.</li>
		<li>Have technician #3 quickly weigh the sample to confirm that an adequate amount of muscle tissue was collected and place the samples on an ice-cold dissection block.</li>
	</ol>
	</li>
	<li>Have technician #3 carefully dissect visible connective tissue and fat from the muscle samples. Have technician #3 prepare the muscle samples for storage according to downstream analyses. For example, immediately place samples in a cryovial and snap freeze in liquid nitrogen, mount and freeze for histology, or place in a cryovial with RNase inhibitor, etc.</li>
	<li>If necessary, repeat the procedure with a second sterilized biopsy needle while maintaining aseptic technique.</li>
</ol>
3. Closure
<ol>
	<li>Have the operator apply direct pressure to biopsy site with sterile gauze and an ice pack for 10-15 min.</li>
	<li>Once hemostasis is achieved, have the operator use alcohol prep pads to remove dried blood from the area around the incision.</li>
	<li>Have technician #1 present a tube of topical surgical adhesive and applicator to the operator. Have the operator assemble the tube of surgical adhesive and applicator without touching the applicator tip or the top of the tube.</li>
	<li>Have the operator pull the incision closed with one hand while applying a single layer of surgical adhesive over the top of the dry incision with the other hand. After the adhesive cures (~ 90 sec), apply tape closuresperpendicular to the incision. Alternatively, close the incision with two sterile 4-0 sutures.</li>
	<li>Have the operator apply direct pressure by placing 3-4 2&#8221; x 2&#8221; non-sterile gauze pads on the incision site and secure with self-adhering adhesive wrap.</li>
	<li>Have the operator provide the subject verbal and written instructions on proper wound care, normal and abnormal reactions, and activity guidelines for the following 1 to 4 days (see online supplement 2).</li>
</ol>

<ol>
	<li>Duchene, G. B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recherches+sur+la+paralysie+musculaire+pseudohypertrophique+ou+paralysie+myo-sclerosique.">Recherches sur la paralysie musculaire pseudohypertrophique ou paralysie myo-sclerosique.</a> Arch Gen Med. 11 (30), (1868).</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.+Determined+by+neutron+activation+analysis+on+needle+biopsy+specimens.">Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens.</a> Scand J Clin Lab Invest (England). 14, (1962).</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=Percutaneous+needle+biopsy+of+skeletal+muscle+in+physiological+and+clinical+research).">Percutaneous needle biopsy of skeletal muscle in physiological and clinical research).</a> Scand J Clin Lab Invest. 35, 609-616 (1975).</li><li>Bergstrom, J., Hultman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+glycogen+metabolism+during+exercise+in+man.">A study of the glycogen metabolism during exercise in man.</a> Scand J Clin Lab Invest. 19, 218-228 (1967).</li><li>Evans, W. J., Phinney, S. D., Young, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction+applied+to+a+muscle+biopsy+maximizes+sample+size.">Suction applied to a muscle biopsy maximizes sample size.</a> Med Sci Sports Exerc. 14, 101-102 (1982).</li><li>Hennessey, J. V., Chromiak, J. A., Della Ventura, S., Guertin, J., MacLean, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+percutaneous+muscle+biopsy+yield+with+a+suction-enhancement+technique.">Increase in percutaneous muscle biopsy yield with a suction-enhancement technique.</a> J Appl Phys. 82, 1739-1742 (1997).</li><li>Tarnopolsky, M. A., Pearce, E., Smith, K., Lach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction-modified+Bergstrom+muscle+biopsy+technique:+experience+with+13,500+procedures.">Suction-modified Bergstrom muscle biopsy technique: experience with 13,500 procedures.</a> Muscl., &amp; Nerve. 43, 717-725 (2011).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+ingestion+influences+skeletal+muscle+cytokine+mRNA+and+plasma+cytokine+levels+after+a+3-h+run.">Carbohydrate ingestion influences skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h run.</a> J Appl Physiol. 94, 1917-1925 (2003).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+carbohydrate+ingestion+on+immune+changes+after+2+h+of+intensive+resistance+training.">Influence of carbohydrate ingestion on immune changes after 2 h of intensive resistance training.</a> J Appl Physiol. 96, 1292-1298 (2004).</li><li>Utter, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+supplementation+and+perceived+exertion+during+prolonged+running.">Carbohydrate supplementation and perceived exertion during prolonged running.</a> Med Sci Sports Exerc. 36, 1036-1041 (2004).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+cytokine+mRNA+changes+after+2.5+h+of+cycling:+influence+of+carbohydrate.">Muscle cytokine mRNA changes after 2.5 h of cycling: influence of carbohydrate.</a> Med Sci Sports Exerc. 37, 1283-1290 (2005).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+quercetin+and+EGCG+on+mitochondrial+biogenesis+and+immunity.">Effects of quercetin and EGCG on mitochondrial biogenesis and immunity.</a> Med Sci Sports Exerc. 41, 1467-1475 (2009).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin's+Influence+on+Exercise+Performance+and+Muscle+Mitochondrial+Biogenesis.">Quercetin's Influence on Exercise Performance and Muscle Mitochondrial Biogenesis.</a> Med Sci Sports Exerc. 42, 338-345 (2010).</li><li>Dumke, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin's+effect+on+cycling+efficiency+and+substrate+utilization.">Quercetin's effect on cycling efficiency and substrate utilization.</a> Appl Physiol Nut Metab. 34, 993-1000 (2009).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin's+influence+on+exercise-induced+changes+in+plasma+cytokines+and+muscle+and+leukocyte+cytokine+mRNA.">Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA.</a> J Appl Physiol. 103, 1728-1735 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline+for+Disinfection+and+Sterilization+in+Healthcare+Facilities.">Guideline for Disinfection and Sterilization in Healthcare Facilities.</a> http://www.cdc.gov/hicpac/Disinfection_Sterilization/toc.html. , (2008).</li><li>Faria, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+onset+and+duration+of+mobilization+affect+the+regeneration+in+the+rat+muscle.">The onset and duration of mobilization affect the regeneration in the rat muscle.</a> Histol Histopath. 23, 565-571 (2008).</li><li>Carlson, B. M., Shepard, B., Komorowski, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histological+study+of+local+anesthetic-induced+muscle+degeneration+and+regeneration+in+the+monkey.">A histological study of local anesthetic-induced muscle degeneration and regeneration in the monkey.</a> J Ortho Res. 8, 485-494 (1990).</li><li>Yagiela, J. A., Benoit, P. W., Buoncristiani, R. D., Peters, M. P., Fort, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+myotoxic+effects+of+lidocaine+with+epinephrine+in+rats+and+humans.">Comparison of myotoxic effects of lidocaine with epinephrine in rats and humans.</a> Anesth Analg. 60, 471-480 (1981).</li><li>Coyle, E. F., Coggan, A. R., Hemmert, M. K., Ivy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+glycogen+utilization+during+prolonged+strenuous+exercise+when+fed+carbohydrate.">Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate.</a> J Appl Physiol. 61, 165-172 (1986).</li><li>Gibala, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+intense+interval+exercise+activates+AMPK+and+p38+MAPK+signaling+and+increases+the+expression+of+PGC-1alpha+in+human+skeletal+muscle.">Brief intense interval exercise activates AMPK and p38 MAPK signaling and increases the expression of PGC-1alpha in human skeletal muscle.</a> J Appl Physiol. 106, 929-934 (1985).</li><li>Phillips, S. M., Tipton, K. D., Ferrando, A. A., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+training+reduces+the+acute+exercise-induced+increase+in+muscle+protein+turnover.">Resistance training reduces the acute exercise-induced increase in muscle protein turnover.</a> Am J Physiol. 276, 118-124 (1999).</li><li>Davis, J. M., Murphy, E. A., Carmichael, M. D., Davis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin+increases+brain+and+muscle+mitochondrial+biogenesis+and+exercise+tolerance.">Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance.</a> Am J Physiol Regul Integr Comp Physiol. 296, 1071-1077 (2009).</li><li>Vissing, K., Andersen, J. L., Schjerling, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+exercise-induced+genes+induced+by+exercise.">Are exercise-induced genes induced by exercise.</a> FASEB J. 19, 94-96 (2005).</li><li>Lundby, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+human+skeletal+muscle:+alternative+normalization+method+and+effect+of+repeated+biopsies.">Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies.</a> Eur J Appl Physiol. 95, 351-360 (2005).</li><li>Coyle, E. F., Coggan, A. R., Hemmert, M. K., Ivy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+glycogen+utilization+during+prolonged+strenuous+exercise+when+fed+carbohydrate.">Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate.</a> J Appl Physiol. 61, 165-172 (1985).</li></ol>

The authors have no conflicts of interest.

This video provides a step-by-step summary of the skeletal muscle biopsy procedure used at the ASU Human Performance Laboratory. This procedure, with small modifications, has been used to collect approximately 1,600 muscle biopsies during the past 13 years. The muscle biopsy samples have provided important data in sports nutrition based investigations, leading to important research discoveries.
There are many critical techniques to be aware of when performing the modified Bergstr&#246;m skelet...

The percutaneous or &#8216;semi-open&#8217; biopsy technique is used to obtain skeletal muscle tissue specimens from human patients and research subjects. Duchenne (1806-1875) is credited as the first to construct a needle with a trocar to obtain skeletal muscle from living subjects through percutaneous biopsy1. In the 1960&#8217;s, Bergstr&#246;m introduced a percutaneous biopsy needle similar to that described by Duchenne2-4. Twenty years later, Evans et al.5 modified the technique by applying suction through the cutting trocar of the Bergstr&#246;m needle. This modification can enhance the yield of tissue 3- to 5-fold6,7 and is employed in clinical and biomedical research settings. This technique has and will continue to foster the diagnosis of myopathies and our understanding of the structure and function of skeletal muscle.
The percutaneous muscle biopsy technique is straightforward. When done correctly, with strict adherence to aseptic technique, the associated risks are minimal. The muscle biopsy procedure is regarded as an investigational tool for research projects. Some academic institutions allow trained faculty researchers with a Ph.D., with direct physician oversight, to obtain muscle biopsies and others require a board-certified physician to perform the technique. The ASU biopsy team has successfully performed over 1,600 muscle biopsies during the past 13 years8-15. The purpose of this video is to describe the modified Bergstr&#246;m needle percutaneous muscle biopsy technique5 to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects.

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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 muscle biopsy procedure as described above allows the researcher to quickly and consistently collect skeletal muscle tissue samples. The typical yield in healthy, athletic subjects is 200 mg or more in a single pass with 3-4 clips. The procedure takes 15-20 min, most of which is spent in preparation for the incision. In exercise-based studies, muscle samples are often taken pre- and post-exercise, with one or two samples collected during recovery. With this design, the hair on the post-exercise (contralateral) thigh ...

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Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#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 ...

human-skeletal-muscle-biopsy-procedures-using-modified-bergstrm

Research

JoVE Journal

Medicine

39.9K Views.  Appalacian State University. The purpose of this video is to present the modified Bergström skeletal muscle biopsy technique on human subjects. 

Video: Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

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

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 ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

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 ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Biochemistry

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 ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

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 ...

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.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

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