Name: induction and assessment of exertional skeletal muscle damage in humans
Uploaded: 2016-12-11T13:00:00
Description: Watch this Scientific Journal Video about Induction and Assessment of Exertional Skeletal Muscle Damage in Humans at JoVE.com

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 ...

<ol>
	<li>Deyhle, M. R., 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=Skeletal+Muscle+Inflammation+Following+Repeated+Bouts+of+Lengthening+Contractions+in+Humans.">Skeletal Muscle Inflammation Following Repeated Bouts of Lengthening Contractions in Humans.</a> Front. Physiol. 6, 424 (2015).</li><li>Hyldahl, R. D., 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=Extracellular+matrix+remodeling+and+its+contribution+to+protective+adaptation+following+lengthening+contractions+in+human+muscle.">Extracellular matrix remodeling and its contribution to protective adaptation following lengthening contractions in human muscle.</a> FASEB J. 29 (7), 2894-2904 (2015).</li><li>Hyldahl, R. D., Olson, T., Welling, T., Groscost, L., Parcell, A. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+rhabdomyolysis+and+acute+renal+failure+in+marathon+runners.">Exertional rhabdomyolysis and acute renal failure in marathon runners.</a> Sports Med. 37 (4-5), 361-363 (2007).</li><li>Seedat, Y. K., Aboo, N., Naicker, S., Parsoo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure+in+the+&amp;#34;Comrades+Marathon&amp;#34+runners.">Acute renal failure in the &amp;#34;Comrades Marathon&amp;#34 runners.</a> Ren. Fail. 11 (4), 209-212 (1989).</li><li>Landau, M. E., Kenney, K., Deuster, P., Campbell, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+rhabdomyolysis:+a+clinical+review+with+a+focus+on+genetic+influences.">Exertional rhabdomyolysis: a clinical review with a focus on genetic influences.</a> J. Clin. Neuromuscul. Dis. 13 (3), 122-136 (2012).</li><li>Warren, G. 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=Role+of+CC+chemokines+in+skeletal+muscle+functional+restoration+after+injury.">Role of CC chemokines in skeletal muscle functional restoration after injury.</a> Am. J. Physiol. Cell Physiol. 286 (5), C1031-C1036 (2004).</li><li>Zhang, 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=CD8+T+cells+are+involved+in+skeletal+muscle+regeneration+through+facilitating+MCP-1+secretion+and+Gr1(high)+macrophage+infiltration.">CD8 T cells are involved in skeletal muscle regeneration through facilitating MCP-1 secretion and Gr1(high) macrophage infiltration.</a> J. Immunol. 193 (10), 5149-5160 (2014).</li><li>Cermak, N. M., Noseworthy, M. D., Bourgeois, J. M., Tarnopolsky, M. A., Gibala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+tensor+MRI+to+assess+skeletal+muscle+disruption+following+eccentric+exercise.">Diffusion tensor MRI to assess skeletal muscle disruption following eccentric exercise.</a> Muscle Nerve. 46 (1), 42-50 (2012).</li><li>Chen, Y. W., Hubal, M. 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>

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.

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	<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...

Watch this Scientific Journal Video about Induction and Assessment of Exertional Skeletal Muscle Damage in Humans at JoVE.com

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 ...

The overall goal of this procedure is to safely induce and quantify exertional skeletal muscle damage to the quadriceps femoris muscles in human subjects using voluntary eccentric contractions. This muscle damage protocol can help researchers answer key questions regarding the mechanisms of muscle adaptation and regeneration following injury. The main advantage of this technique it that we can study the processes of muscle adaptation in human subjects, eliminating the need for interspecies translation of the results.While this method can be used to study muscle damage in the context of exercise, it can also be used to study damage caused by overt injury. Demonstrating the procedure will be Jake Sorensen, a PhD student from my laboratory. To create the isokinetic strength test protocol, begin by opening the dynamometer control software on the computer.Select new protocol from the dropdown list, then on the unilateral tab, program one set of three repetitions at a speed of 60 degrees per second for both the away and toward contractions by entering one into the number sets field, typing three in the end by reps row, and typing 60 in both the speed away and speed toward rows. Next, create an isometric strength test with one set of three isometric contractions lasting five seconds each at a joint angle of 70 degrees by typing a one into the positions field on the unilateral tab and typing a three into the end by reps row under the position one column. Then select 70 in the row labeled angle.Next, program the muscle damaging eccentric exercise protocol by clicking the unilateral tab and typing 10 into the number of sets field. In the first column, type 10 in the row labeled end by reps. Then type 180 in the next row, labeled speed away and 120 in the row labeled speed toward.Lastly, in the row labeled torque, type 200 to 600 depending on the anticipated strength of the subject, and enter 60 into the field labeled rest time in seconds. Begin by making a visual analog scale by drawing a 100 millimeter line horizontally across a page. On the left hand side of the line, indicate no soreness, and on the right hand side of the line, indicate extreme soreness.Next, escort the subject into the testing room, and instruct them to do two body weight squats to collect baseline soreness measurements. Perform both squats with arms help straight out in front of the shoulders, and feet shoulder width apart, in which proper depth is reached once the upper legs are parallel to the floor. Then, 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.Finally quantify the subjects level of soreness by measuring the distance in millimeters from the no soreness end of the visual analog scale to the subject's mark. After a brief warmup on a cycle ergometer, ask the subject to sit in the chair of the dynamometer. Adjust the dynamometer position settings such that the rotational axis of the knee aligns with that of the lever arm, and secure the leg to the shaft arm.Record these settings for repeatable set up in the future. Stabilize the subject with shoulder, hip, ankle, and thigh straps. Next, inform the subject that they will be contracting against the lever arm that will remain stationary.You should instruct the subject according to the prompts on the computer screen to complete three contractions lasting five seconds each. Start the isometric protocol on the computer. Set the ROM limits, calibrate the dynamometer, and allow the subject to practice the test two to three times with submaximal effort in order to familiarize the subject to the test.Once the subject is ready, ask them to cross their arms across their chest. Start the test, and verbally encourage the subject to provide maximal effort for each contraction. Record baseline measurements of torque, power, and work.Allow the subject two to three minutes to rest, and then load the isokinetic strength test protocol on the computer. Inform the subject that they will complete three extension and flexion contractions against the lever arm that will move at a fixed rate. Let them know that the lever arm will stop when they reach the end of the range of motion, and the goal is to contract as forcefully as possible throughout the entire range of motion for each contraction.After a few submaximal practice tests to familiarize the subject, ask them to cross their arms across their chest, and start the test. Verbally encourage the subject to provide maximal effort, and to breathe normally. Record baseline measurements of torque, power, and work.One to three days after collecting the isometric and isokinetic baseline measurements, have the subject return to the lab to complete the muscle damaging eccentric exercise protocol. After a warmup, orient and secure the subject in the dynamometer using the previously recorded adjustment settings and load the exercise protocol. Instruct the subject to contract against the lever arm to initiate the movement of the shaft arm for each contraction.Then, explain that as the shaft arm moves toward them at a fixed rate, their goal is to contract against it as forcefully as possible. When the subject is ready, start the first set of contractions by pushing the start button on the screen, and as they perform the eccentric contractions, verbally encourage them to maintain maximal effort for each contraction. Finally, immediately after the muscle damaging exercise, repeat both isokinetic and isometric strength tests as well as at 24, 48, 72, and 96 hours after.Additionally, repeat the soreness measurement at 24, 48, 72, and 96 hours after the exercise test. Results from seven untrained young men show that compared to pre-exercise, isometric and isokinetic force was decreased out to 24 and 48 hours post exercise, respectively, and returned similar to pre-exercise values thereafter. Conversely, soreness was significantly increased at 24, 48, and 72 hours after exercise.Once mastered, this technique can be done safely and reliably if it's performed properly. Remember that it's important to consider the physical limitations of each subject and adapt the parameters accordingly. The joint angle parameters in the protocol should be reduced for individuals with limited range of motion and the target force should be reduced for weaker individuals.Following this procedure, other methods like obtaining a blood or muscle sample can be performed in order to answer additional questions related to the cellular and molecular responses to damage. After watching this video, you should have a good understanding of how to safely and ethically induce and quantify exertional muscle damage in human subjects. Lastly, don't forget that this protocol has the small potential to cause rhabdomyolysis.Subjects should be screened for signs and symptoms of rhabdomyolysis, which include dark brown urine, nausea, and extreme muscle weakness during the followup visits.

induction-assessment-exertional-skeletal-muscle-damage

Research

JoVE Journal

Medicine

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

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, ...

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 ...

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 ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...