The authors have no acknowledgements.

The following procedures are in accordance with the standards of the Brigham Young University Institutional review board (IRB).
1. Prepare the Contraction Protocols
NOTE: The following protocol instructions are based on the Biodex Advantage software. Navigating the software and operating the dynamometer will be different if different systems are used.
<ol>
	<li>Isokinetic Strength Test Protocol
	<ol>
		<li>To make the isokinetic protocol, open dynamometer control software on the computer and select the &#34;Protocol&#34; tab and then select the &#34;Record&#34; tab on the toolbar on the top of the screen. Select &#34;New Protocol&#34; from the dropdown list.</li>
		<li>In the &#34;Study Type&#34; box, select the &#34;Test&#34; option.</li>
		<li>Select the following parameters from the dropdown menus: Under the &#34;Mode&#34; menu select Isokinetic. Under the &#34;Joint&#34; menu select knee. Under the &#34;Pattern&#34; menu select extension/flexion. Select concentric contractions for both extension and flexion movements. Select &#34;reps&#34; under the &#34;End By&#34; dropdown menu.</li>
		<li>On the &#34;Unilateral&#34; tab program 1 set of 3 repetitions at a speed of 60&#176;/sec for both the away and toward contractions. To do this, enter &#34;1&#34; into the &#34;# sets&#34; field and type &#34;3&#34; in the &#34;End By Reps&#34; row. Then type 60 in both the &#34;Speed Away&#34; and &#34;Speed Toward&#34; rows.</li>
		<li>Use 90&#176; as the anatomical reference point and set range of motion limits to approximately 10&#176; away and 110&#176; towards (0&#176; = full extension, 135&#176; = full flexion).To do this, select the &#34;set ROM&#34; button on the left side of the screen. Calibrate the lever arm so that 90&#176; is parallel to the floor (as indicated using the lever arm angle indicator provided on the computer screen). Then using 90&#176; as the anatomical reference point, set the range of motion limits to approximately 10&#176; away and 110&#176; towards (0&#176; = full extension, 135&#176; = full flexion). 
		NOTE: The joint angle settings given here are preferred, however these settings should be modified on a case-by-case basis especially when working with subjects with a fixed flexion deformity or other limitation.</li>
		<li>Name the protocol in the &#34;Description&#34; field on the bottom left of the screen and select the save button on the top menu bar.</li>
	</ol>
	</li>
	<li>Isometric Strength Test Protocol
	<ol>
		<li>Repeat steps 1.1.1 to begin making the isometric protocol.</li>
		<li>Select the &#34;Test&#34; option from the &#34;Study Type&#34; box.</li>
		<li>Select the following parameters from the dropdown menus: Under the &#34;Mode&#34; menu select &#34;isometric&#34;. Under the &#34;Joint&#34; menu select &#34;knee&#34;. Under the &#34;Pattern&#34; menu select &#34;extension/flexion&#34;. Under the &#34;Contraction Direction&#34; menu select &#34;away&#34;. In the &#34;End By&#34; menu select &#34;reps&#34;.</li>
		<li>Program a test with 1 set of 3 isometric contractions lasting 5 sec each at a joint angle of 70&#176;. To do this, type &#34;1&#34; into the &#34;Positions&#34; field on the &#34;Unilateral&#34; tab. Under the position #1 column, type &#34;3&#34;in the &#34;End by Reps&#34; row. In the row labeled &#34;Angle&#34; select &#34;70&#34;.</li>
		<li>Select 90&#176; as the anatomical reference point and an away limit to 70&#176;.</li>
		<li>Name the protocol in the &#34;Description&#34; field on the bottom left of the screen and select the save button on the top menu bar.</li>
	</ol>
	</li>
	<li>Muscle Damaging Eccentric Exercise Protocol
	<ol>
		<li>To make the eccentric exercise protocol, open dynamometer control software on the computer and select the &#34;Protocol&#34; tab. Then select the &#34;Record&#34; tab on the toolbar on the top of the screen. Now click on &#34;New Protocol&#34; from the dropdown list.</li>
		<li>Select the &#34;Exercise&#34; option from the &#34;Study Type&#34; box.</li>
		<li>Select the following parameters from the dropdown menus: Under the &#34;Mode&#34; menu select &#34;isokinetic&#34;. Under the &#34;Joint&#34; menu select &#34;knee&#34;. Under the &#34;Pattern&#34; menu select &#34;Extension/Flexion&#34;. Under the &#34;Contraction&#34; menu select &#34;Con/ECC&#34;.</li>
		<li>On the &#34;Unilateral&#34; tab, program 10 sets of 10 repetitions at speeds of 180&#176;/sec away and 120&#176;/sec towards with 60 sec rest between sets.
		<ol>
			<li>Type &#34;10&#34; into the &#34;# Sets&#34; field.</li>
			<li>In the first column (labeled #1), type &#34;10&#34; in the row labeled &#34;End by Reps&#34;. Then type &#34;180&#34; in the next row labeled &#34;Speed Away&#34;. In the next row labeled &#34;Speed Toward&#34;, type &#34;120&#34;. In the row labeled &#34;Torque&#34;, type 200 to 600 depending on the anticipated strength of the subject.</li>
			<li>Repeat step 1.3.4.2 for sets #2 through #10.</li>
			<li>Type 60 in the &#34;Rest Time in secs&#34; field.</li>
		</ol>
		</li>
		<li>Type 90&#176; into the &#34;Anatomical Reference&#34; field.</li>
		<li>Select the &#34;Set ROM&#34; button on the left side of the screen and set range of motion limits to 40&#176; away and 110&#176; towards (0&#176; = full extension, 135&#176; = full flexion). These settings provide the range of motion through which the subject must provide resistance against the arm of the dynamometer. As a safety precaution, the dynamometer will stop moving if the subject stops providing resistance within this specified range of motion.
		<ol>
			<li>To set the range of motion, select the &#34;set ROM&#34; button on the left side of the screen. Calibrate the lever arm so that 90&#176; is parallel to the floor (as indicated using the lever arm angle indicator provided on the computer screen). Then using 90&#176; as the anatomical reference point, set the range of motion limits to 40&#176; away and 110&#176; towards (0&#176; = full extension, 135&#176; = full flexion). 
			NOTE: These joint angle settings are suggestions for a typical healthy subject. These should be modified to fit the abilities from one individual to another.</li>
		</ol>
		</li>
		<li>Name the protocol in the &#34;Description&#34; field and select the &#34;Save&#34; button at the top tool bar.</li>
	</ol>
	</li>
</ol>
2. Baseline Measurements
<ol>
	<li>Soreness Measurements
	<ol>
		<li>Make a visual analog scale by drawing a 100 mm line horizontally across a page.</li>
		<li>On the left-hand side of the line indicate &#34;No soreness&#34; and on the right-hand side of the line indicate &#34;Extreme soreness&#34; (Figure 1). This method is commonly used to assess delayed-onset muscle soreness1,10,11.</li>
		<li>Instruct the subject to do two body-weight squats. Perform both squats with arms held straight out in front of the shoulders and feet shoulder-width apart. Proper depth is reached when the upper legs are parallel to the floor before returning to the start position.</li>
		<li>Ask the subject to indicate the intensity of the soreness he or she felt in the quadriceps femoris muscles during the squats by drawing a vertical line at the appropriate spot on the visual analog scale.</li>
		<li>Quantify the subject&#39;s level of soreness by measuring the distance in millimeters from the no-soreness end of the visual analog scale to subject&#39;s mark.</li>
	</ol>
	</li>
	<li>Serum Creatine Kinase 
	CAUTION: Wear protective gloves before handling blood and dispose of needles immediately after use in a sharps container as per hospital or university policy.
	<ol>
		<li>Follow standardized phlebotomy procedures to withdraw blood from the antecubital region of the subject&#39;s arm under fasting conditions to obtain 5 to 6 ml of blood12.</li>
		<li>Centrifuge the blood sample at 15 x g for 15 min at 3 &#176;C to separate serum from blood cells.</li>
		<li>Using a transfer pipette, transfer the serum sample into a microcentrifuge tube labeled appropriately for subject and time point.</li>
		<li>Store the sample at -80 &#176;C for later use.</li>
		<li>Assay the serum samples for creatine kinase activity using a commercially available kit according to manufacturer&#39;s protocol. Alternatively, the serum samples can be transported to a local medical diagnostic laboratory for analysis.</li>
	</ol>
	</li>
	<li>Subject Preparation
	<ol>
		<li>Forty-eight hours prior to the subject&#39;s visit, instruct them to avoid strenuous or unaccustomed exercise, non-steroidal anti-inflammatory drugs and alcohol.</li>
		<li>When the subject arrives, collect weight, height, sex, and leg dominance information prior to setup. Leg dominance can be determined by asking the subject with which leg they would choose to kick a ball.</li>
		<li>Have the subject warm up at 50 to 150 W for 10 min on a bicycle ergometer before performing the strength tests.</li>
		<li>After the warm-up ask the subject to sit in the chair of the dynamometer. Adjust the tilt, height, depth, recline, and rotation of the seat and shaft arm to fit the subject. Use the lateral femoral condyle as a reference and align rotational axis of the knee with that of the dynamometer shaft arm. Adjust the shaft arm length so that it is just proximal to the medial malleolus, and secure with strap. Subject should be able to plantar flex comfortably with full range of motion.</li>
		<li>Leave sufficient space (25 mm) between the front edge of the seat and the back of the leg to allow maximal flexion of the knee joint without pinching or discomfort.</li>
		<li>Stabilize subject with shoulder, hip, ankle and thigh straps. To isolate the knee extensors and avoid ancillary muscle involvement, it is essential that the subject be securely stabilized.</li>
		<li>Have the subject perform 3-5 practice extension/flexion maneuvers of the knee, monitor range of motion and signs of discomfort that may exist prior to beginning the contraction protocol, and adjust as needed.</li>
		<li>Now that the adjustable components of the dynamometer are properly fitted to the subject, record the seat position settings and shaft arm position for repeatable setup on subsequent visits. 
		NOTE: It is important that the subject is safe and comfortable. The dynamometer is equipped with an emergency stop button for both the subject and operator in the case of an emergency.</li>
	</ol>
	</li>
	<li>Force Measurement
	<ol>
		<li>After the subject is properly oriented in the seat and the isometric strength test protocol is loaded, instruct the subject on how to perform the test.
		<ol>
			<li>Tell the subject that he or she will be contracting against the lever arm that will stay stationary. Tell the subject to follow the prompts of the computer screen doing 3 contractions lasting 5 sec each.</li>
			<li>Tell the subject to maintain maximal effort for each contraction. Tell the subject to breathe during the contractions. Ask the subject to cross their arms across the chest during the test.</li>
			<li>Start the isometric protocol and allow the subject to practice the test with submaximal effort. This practice trial will familiarize the subject to the test as well as provide a test-specific warm-up. Allow the subject to practice the test 2 to 3 times.</li>
		</ol>
		</li>
		<li>Once the subject is ready, start the test and verbally encourage the subject to provide maximal effort for each contraction.</li>
		<li>Record torque, power, and work. The software also calculates the coefficient of variance (a statistical representation of test validity based on reproducibility of performance, lower values indicate higher reproducibility). If the coefficient of variance exceeds 15%, repeat the test. Repeat the test up to 4 times. If the subject is unable to produce a coefficient of variance less than 15% after 4 attempts use the data from the set that yielded the lowest coefficient of variance value.</li>
		<li>Allow the subject two to three minutes to rest, and then load the isokinetic protocol to obtain isokinetic strength data.</li>
		<li>After the subject is secured in the seat, instruct on how to perform the isokinetic test.
		<ol>
			<li>Tell the subject that they will be doing three extension and flexion contractions against the lever arm that will move at a fixed rate. Tell the subject the goal is to contract as forcefully as possible throughout the entire range of motion for each contraction.</li>
			<li>Tell them that the lever arm will stop when they reach the end of the range of motion. Ask the subject to cross his arms across the chest during the test. Remind the subject not to hold their breath during the contractions 
			NOTE: While the data gathered from the flexion component of the contraction may not be of interest to the researcher, subjects who provide maximal effort for both the flexion and extension maneuvers tend to yield better force consistency during the extension maneuver.</li>
		</ol>
		</li>
		<li>Start the test and verbally encourage the subject to provide maximal effort.</li>
		<li>Record torque, power, and work. The software also calculates the coefficient of variance (a statistical representation of test validity based on reproducibility of performance, lower values indicate higher reproducibility). If the coefficient of variance exceeds 15%, repeat the test.
		<ol>
			<li>Repeat the test up to 4 times. If the subject is unable to produce a coefficient of variance less than 15% after 4 attempts, use the data from the set that yielded the lowest coefficient of variance value.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Damage Induction
<ol>
	<li>One to three days after the baseline measurements of Part 2, have the subject return to the lab for the damage induction portion of the protocol</li>
	<li>Have the subject warm-up on a cycle ergometer for 10 min at 50 to 150 W.</li>
	<li>After the warm-up, orient and secure the subject in the dynamometer using the adjustment settings recorded in step 2.3.7, load the muscle damaging eccentric exercise protocol and instruct the subject on how to properly perform the exercise.
	<ol>
		<li>Tell the subject that they will contract against the lever arm to initiate the movement of the shaft arm for each contraction. Tell the subject that as the shaft arm moves toward them at a fixed rate, their goal is to contract against it as forcefully as possible.</li>
		<li>Allow the subject to grasp the handgrips located on either side of the seat.</li>
		<li>Tell the subject to breathe during the contractions; exhale during the eccentric phase.</li>
	</ol>
	</li>
	<li>When the subject is ready, start the first set of contractions by pushing the start button on the screen.</li>
	<li>Verbally encourage the subject to maintain maximal effort for each contraction.</li>
	<li>Follow the prompts on the screen that will guide the subject through 10 sets of 10 repetitions with a 1 min rest period between sets.</li>
	<li>Once 10 sets are completed allow the subject 1 to 5 min to rest before repeating the protocol. 
	NOTE: The maximum number of sets available under the eccentric protocol is 10 however this may vary depending on the dynamometer and software used. The rest provides time for the operator to reload the protocol if necessary.</li>
	<li>Repeat the procedure two more times for a total of 300 predominantly eccentric contractions.</li>
</ol>
4. Muscle Damage Assessment
<ol>
	<li>Repeat both Isokinetic and isometric strength tests as described in 2.4 immediately after the damaging exercise and at 24, 48, 72, and 96 hr after.</li>
	<li>Repeat the soreness measurement as described in part 2.1 at 24, 48, 72, and 96 hr after damaging exercise. Be sure to use a new visual analog scale for each assessment so that the subject cannot see their previous responses.</li>
	<li>Repeat the blood draw as described in part 2.2 for the creatine kinase assay at 48 and 120 hr after damaging exercise, or at time points dictated by the experimental protocol.</li>
	<li>Plot the creatine kinase, strength and soreness data on the y-axis of a graph against time on the x-axis.</li>
	<li>Observe how these variables deviate from and return toward baseline values in the days following exercise, indicating damage and repair, respectively.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+activity+is+differentially+affected+by+contraction+mode+in+human+muscle+following+a+work-matched+bout+of+exercise.">Satellite cell activity is differentially affected by contraction mode in human muscle following a work-matched bout of exercise.</a> Front. Physiol. 5, 485 (2014).</li><li>Clarkson, P. M., Kearns, A. K., Rouzier, P., Rubin, R., Thompson, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+creatine+kinase+levels+and+renal+function+measures+in+exertional+muscle+damage.">Serum creatine kinase levels and renal function measures in exertional muscle damage.</a> Med. Sci. Sports Exerc. 38 (4), 623-627 (2006).</li><li>Clarkson, P. 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J., Hoffman, E. P., Thompson, P. D., Clarkson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+responses+of+human+muscle+to+eccentric+exercise.">Molecular responses of human muscle to eccentric exercise.</a> J. Appl. Physiol. 95 (6), 2485-2494 (2003).</li><li>Stasinger, S. K., Di Lorenzo, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phlebotomy Textbook. , 188-203 (2011).</li><li>Hubal, M. J., Chen, T. C., Thompson, P. D., Clarkson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+gene+changes+associated+with+the+repeated-bout+effect.">Inflammatory gene changes associated with the repeated-bout effect.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 294 (5), R1628-R1637 (2008).</li><li>Stupka, N., Tarnopolsky, M. A., Yardley, N. J., Phillips, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+adaptation+to+repeated+eccentric+exercise-induced+muscle+damage.">Cellular adaptation to repeated eccentric exercise-induced muscle damage.</a> J. Appl. Physiol. 91 (4), 1669-1678 (2001).</li><li>Smith, L. 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=Changes+in+serum+cytokines+after+repeated+bouts+of+downhill+running.">Changes in serum cytokines after repeated bouts of downhill running.</a> Appl. Physiol. Nutr. Metab. 32 (2), 233-240 (2007).</li><li>Marqueste, T., Giannesini, B., Fur, Y. L., Cozzone, P. J., Bendahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+MRI+analysis+of+T2+changes+associated+with+single+and+repeated+bouts+of+downhill+running+leading+to+eccentric-induced+muscle+damage.">Comparative MRI analysis of T2 changes associated with single and repeated bouts of downhill running leading to eccentric-induced muscle damage.</a> J. Appl. Physiol. 105 (1), 299-307 (2008).</li><li>Crameri, R. M., 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=Myofibre+damage+in+human+skeletal+muscle:+effects+of+electrical+stimulation+versus+voluntary+contraction.">Myofibre damage in human skeletal muscle: effects of electrical stimulation versus voluntary contraction.</a> J. Physiol. 583 (Pt 1), 365-380 (2007).</li><li>Yu, J. G., Malm, C., Thornell, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eccentric+contractions+leading+to+DOMS+do+not+cause+loss+of+desmin+nor+fibre+necrosis+in+human+muscle.">Eccentric contractions leading to DOMS do not cause loss of desmin nor fibre necrosis in human muscle.</a> Histochem. Cell Biol. 118 (1), 29-34 (2002).</li><li>Jamurtas, A. Z., 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=Comparison+between+leg+and+arm+eccentric+exercises+of+the+same+relative+intensity+on+indices+of+muscle+damage.">Comparison between leg and arm eccentric exercises of the same relative intensity on indices of muscle damage.</a> Eur. J. Appl. Physiol. 95 (2-3), 179-185 (2005).</li></ol>

The authors have nothing to disclose.

Several steps are critical to obtaining the desired results of this protocol. First, subjects must be adequately familiarized to the contraction protocols, particularly the force measurements. Be sure that the subject understands exactly what they are expected to do and give them an opportunity to practice the strength tests prior to data collection. Subjects who are not adequately familiarized with these procedures may show a learning curve over the days following the damage induction. This can be a confounding variable...

The overall goal of this procedure is to induce exertional damage to the quadriceps femoris muscles using voluntary lengthening (eccentric) contractions in human subjects.
Contraction-induced skeletal muscle damage is a common consequence of exercise that is marked by delayed onset muscle soreness1, transient strength loss, and elevated muscle-specific enzymes in the blood2. Exertional muscle damage is most pronounced following exercise to which the subject is unaccustomed, particularly when eccentric contractions are involved3. Exertional muscle damage is typically benign. Soreness subsides, and both serum proteins and strength typically return to pre-damage levels within a few days to weeks after the damaging insult. In extreme cases, exertional muscle damage can lead to a life-threatening syndrome know as rhabdomyolysis. However, exertional muscle damage is usually insufficient to cause clinical rhabdomyolysis in healthy individuals4 in the absence of compounding factors including heat stress, dehydration5, infection6 or rare genetic predispositions7. 
Contraction-induced muscle damage is typically less severe than toxin-induced or freezing-induced injury, methods often used in rodent studies8,9. Yet, contraction-induced injury provides a useful method to study the muscle damage response with notable advantages. First, it is a safe and ethical method for use with human subjects1-3. Thus, interspecies translation of the results is not needed as data can be obtained directly from human subjects. Moreover, translating data obtained from rodent studies is very difficult given that the severity of injury seen in the rodent injury models exceeds the level of damage that would be ethical to induce in human subjects. Second, contraction-induced damage is commonly experienced and a natural process of exercise. Therefore, this mode of damage induction is useful for studying muscle damage in the context of exercise, adaptation to exercise as well as overt muscle injury. Here we describe a safe and reliable method to induce and evaluate skeletal muscle damage using lengthening contractions in humans.

<table border="0" cellpadding="0" cellspacing="0" width="924">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Biodex Dynomometer</td>
			<td style="width:167px;">Biodex Medical Systems</td>
			<td style="width:119px;">850-000</td>
			<td style="width:423px;">Other models are available and should produce similar results</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Creatine Kinase kit</td>
			<td style="width:167px;">Sigma-Aldrich&#160;</td>
			<td>MAK116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serum Vacutainers</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:119px;">367812</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Winged safety push button blood collection set</td>
			<td style="width:167px;">BD Bioscience</td>
			<td style="width:119px;">367338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
			<td style="width:167px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">V5007</td>
			<td>We use the 2mL vials to store serum aliquots</td>
		</tr>
	</tbody>
</table>

induction and assessment of exertional skeletal muscle damage in humans

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

Using the methods presented here, baseline soreness, serum creatine kinase activity, and strength (isometric and isokinetic) measurements were taken in 7 untrained young men. The following day, the subjects underwent the muscle damaging eccentric contraction protocol described above. To provide indices of muscle damage, follow up assessments of strength, soreness and serum creatine kinase activity were made. Strength was measured immediately after as well as 24, 48, 72, and 96 hr after ex...

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Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

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

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

induction-assessment-exertional-skeletal-muscle-damage

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JoVE Journal

Medicine

7.4K Views.  Brigham Young University. This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Video: Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

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 ...

Skeletal Muscle Gender Dimorphism from Proteomics

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

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

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

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

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 ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

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

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

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

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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 ...

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 ...