Supported by Merit Review #BX001026 from the Department of Veterans Affairs (LSQ), and use of resources and facilities at VA Puget Sound Health Care System, the Transgenic Resource Core at the University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging (NIA #5P30AG-013280), and the University of Washington Diabetes Endocrinology Research Center (NIH #P30 DK-17047). We thank Cynthia Pekow DVM and Kari L. Koszdin DVM, VA Puget Sound, provided helpful comments on the manuscript.

The procedure described here was approved by the VA Puget Sound Institutional Animal Care and Use Committee, and complies with the ILAR Guide for the Care and Use of Laboratory Animals.
1. Experimental Preparation
<ol>
	<li>Decide beforehand if blood or tissue collection soon after exercise is necessary, and if so, determine the post-exercise interval for conduction of such procedures. Examples include determination of exercise induced hormone or cytokine release into the circulation (necessitating blood collection), determination of exercise induced expression of mRNA species of interest (necessitating euthanasia followed by tissue collection). If immediate post exercise blood or tissue collection is indicated, have materials for these procedures on hand in the procedure room. Receive institutional review board approval and perform according to regulatory guidelines. Not all experiments require postexercise blood or tissue collection; a simple comparison of running endurance may be all that is needed.</li>
	<li>Decide in advance on experimental groups and postexercise procedures for mice (see description above).</li>
	<li>Place the treadmill on a sturdy table in a quiet room dedicated during the running trials to this procedure.</li>
	<li>Set the treadmill for mouse-sized lanes and at an inclination appropriate to the protocol. The inclination of +5o is used here to obviate muscle damage due to eccentric exercise (lengthening the muscle at the same time as contraction) that occurs during downhill running.</li>
	<li>Place an absorbent pad below the treadmill belt to catch feces.</li>
</ol>
2. Acclimation of Mice to the Treadmill
Decide if a &#8220;sedentary&#8221; (no exercise) group of mice is necessary, and expose sedentary mice to the treadmill without running as outlined in Steps 2.1 - 2.5. Determine if 2 or 4 experimental groups are required, e.g.,&#160;Sedentary/Exercise (2 groups); Exercised Controls/Exercised Treatment (2 groups, no sedentary animals); or, Sedentary/Exercised x Control/Treatment (4 groups). The &#8220;treatment&#8221; may be a transgenic or knockout genotype, a pharmacological regimen, special diet, or other experimental intervention.
<ol>
	<li>Allow the mouse to acclimate to the procedure room in its transported home cage, with cage mates, for 1 - 2 hr.</li>
	<li>Select a single mouse and record tag number; weigh mouse and record weight.</li>
	<li>Place mouse in the treadmill without the motor turned on for acclimation, about 5 min. Turn power on, without the treadmill belt running, and let the mouse acclimate to the machine noise for another 5 min.</li>
	<li>Remove mice in the &#8220;sedentary&#8221; groups (if used; see Note 1) from the treadmill at this point and return them to their cage or conduct post-exercise procedures as per the experimental design (see Note 2).</li>
</ol>
3. Starting the Run-to-Exhaustion test
<ol>
	<li>After acclimation, turn the belt to a low speed (10 m/min) when the animal is exploring the belt and not on the platform.</li>
	<li>Gently tap or lift the hindquarters of the mouse with a tongue depressor, one to three times, to encourage reluctant animals to stay on the treadmill and run. 
	NOTE: Most mice will run readily, but they sometimes stop for short periods. When this occurs, use the tongue depressor to gently tap or lift the rear of the animal to encourage resumption of running. Often, the mice do not need to be touched with the tongue depressor; noticing the hand with the tongue depressor coming towards them causes the mice to run some more.</li>
	<li>Start a laboratory timer once the mouse is running at the initial speed of 10 m/min.</li>
	<li>Turn the speed up slowly when the mouse is at the top (uphill end) of the belt, at increments of 1 m/min, about every 2 min.</li>
	<li>Turn the speed down to the previous setting if the mouse repeatedly runs in short bursts and not at a steady pace.</li>
	<li>Determine the maximal sustained running speed of each mouse (usually 14 - 17 m/min for C57BL/6 mice) by adjusting the speed and watching the mouse. Record this speed. 
	NOTE: The location of the animal on the treadmill cues the investigator as to the willingness of the animal to run at a higher speed. If the mouse is at the far (uphill) end and about to run into the wall, it is ready to run at a higher speed. If the mouse is running at a steady pace in the middle of the treadmill it may be at its maximal speed, or may need further adjustment, depending on the quality of the run.</li>
</ol>
4. Endpoints and Mice that Stop
<ol>
	<li>Use the tongue depressor to gently tap or lift the hindquarters of the animals that stop, to encourage resumption of running (see Note 3.2). Do not stop the timer for temporary interruptions in running.</li>
	<li>Identify and remove mice that refuse to run . Note this on the data sheet. Depending on your experimental design, mice that refuse can be retested on a different day 
	NOTE: Some mice will completely refuse to run. Such mice will run in short bursts, then stop to groom or &#8220;pedal&#8221;, that is, sit on the platform and use their front legs on the belt. The posture of these mice is hunched, with all four feet planted under the animal to keep from being pushed back onto the treadmill by the tongue depressor. Occasionally a mouse becomes aggressive towards the tongue depressor when unwilling to run, biting the tongue depressor or climbing up it as a way of escape. It is clear within the first 5&#160;min&#160;of the trial whether the mouse will, or will not, run. Remove mice that refuse to run from the analysis, but do not count them as &#8220;sedentary&#8221; mice.</li>
	<li>Identify exhausted mice and stop the timer. 
	NOTE: Exhaustion is defined by three successive stops and refusal to resume running despite gentle encouragement, plus physical signs of exhaustion such as labored breathing and splayed posture. Mice recover from exhaustion within 30 to 60 min.</li>
	<li>Record the total amount of time spent running at all speeds.</li>
	<li>Depending on the experimental design, either return the mouse is to its cage, or conduct immediate postexercise procedures such as blood or tissue collection as per institutional guidelines.</li>
	<li>Wipe the belt clean with a germicidal towelette after testing each individual mouse so that the scent of the previous mouse does not influence behavior of subsequent mice tested.</li>
	<li>Test the next mouse. Use a fresh tongue depressor for each mouse. Mice that reside in the same cage can be tested sequentially on the same day without additional acclimation to the procedure room.</li>
</ol>

<ol><li>Hill, J. A., Wyatt, H. R., Reed, G. W., Peters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obesity+and+the+environment%3A+where+do+we+go+from+here%3F%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Obesity and the environment: where do we go from here?</a> Science. 299 (5608), 853-855 (2003).</li>
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<li>Van Gaal, L. F., Mertens, I. L., De Block, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+linking+obesity+with+cardiovascular+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanisms linking obesity with cardiovascular disease.</a> Nature. 444 (7121), 875-880 (2006).</li>
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<li>LeBrasseur, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myostatin+inhibition+enhances+the+effects+of+exercise+on+performance+and+metabolic+outcomes+in+aged+mice%5BTitle%5D)+AND+%22J.+Gerontol.+A+Biol.+Med.+Sci%22%5BJournal%5D)">Myostatin inhibition enhances the effects of exercise on performance and metabolic outcomes in aged mice.</a> J. Gerontol. A Biol. Med. Sci. 64 (9), 940-948 (2009).</li>
<li>Burch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electric+pulse+stimulation+of+cultured+murine+muscle+cells+reproduces+gene+expression+changes+of+trained+mouse+muscle%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle.</a> PLoS ONE. 5 (6), e10970(2010).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+biogenesis+and+peroxisome+proliferator-activated+receptor-%CE%B3+coactivator-1%CE%B1+%28PGC-1%CE%B1%29+deacetylation+by+physical+activity%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Mitochondrial biogenesis and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) deacetylation by physical activity.</a> Diabetes. 60 (1), 157-167 (2011).</li>
<li>Knab, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repeatability+of+exercise+behaviors+in+mice%5BTitle%5D)+AND+%22Physiol.+%26+Behavior%22%5BJournal%5D)">Repeatability of exercise behaviors in mice.</a> Physiol. & Behavior. 98 (4), 433-440 (2009).</li>
<li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-15+Receptor+deletion+results+in+circadian+changes+of+locomotor+and+metabolic+behavior%5BTitle%5D)+AND+%22J.+Mol.+Neurosci%22%5BJournal%5D)">IL-15 Receptor deletion results in circadian changes of locomotor and metabolic behavior.</a> J. Mol. Neurosci. 41 (2), 315-321 (2010).</li>
<li>Wu, X., Hsuchou, H., Kastin, A. J., Rood, J. C., Pan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essential+role+of+interleukin-15+receptor+in+normal+anxiety+behavior%5BTitle%5D)+AND+%22Brain+Behav.+Immun%22%5BJournal%5D)">Essential role of interleukin-15 receptor in normal anxiety behavior.</a> Brain Behav. Immun. 24 (8), 1340-1346 (2010).</li>
<li>Pistilli, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+IL-15+receptor+%CE%B1+alters+the+endurance%2C+fatigability%2C+and+metabolic+characteristics+of+mouse+fast+skeletal+muscles%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Loss of IL-15 receptor α alters the endurance, fatigability, and metabolic characteristics of mouse fast skeletal muscles.</a> J. Clin. Invest. 121 (8), 3120-3132 (2011).</li>
<li>Quinn, L. S., Anderson, B. G., Conner, J. D., Wolden-Hanson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-15+overexpression+promotes+endurance%2C+oxidative+energy+metabolism%2C+and+muscle+PPAR%CE%B4%2C+SIRT1%2C+PGC-1%CE%B1%2C+and+PGC-1%CE%B2+expression+in+male+mice%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">IL-15 overexpression promotes endurance, oxidative energy metabolism, and muscle PPARδ, SIRT1, PGC-1α, and PGC-1β expression in male mice.</a> Endocrinology. 154 (1), 232-245 (2013).</li>
<li>Quinn, L. S., Anderson, B. G., Conner, J. D., Wolden-Hanson, T., Marcell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-15+is+required+for+post-exercise+induction+of+the+pro-oxidative+mediators+PPARdelta+and+SIRT1%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">IL-15 is required for post-exercise induction of the pro-oxidative mediators PPARdelta and SIRT1.</a> Endocrinology. , (2013).</li>
<li>Ward, J. M., Anver, M. R., Mahler, J. F., Devor-Henneman, D. E. Chapter 13, Pathology of mice commonly used in genetic engineering (C57BL/6; 129; B6,129; and FVB/N). <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWard%2C+JM+%22The+Pathology+of+Genetically+Engineered+Mice%22">The Pathology of Genetically Engineered Mice</a>. Ward, J. M., Mahler, J. F., Maronpot, R. R., Sundberg, J. P. , Iowa State University Press. Ames, IA, Chapter. 161-179 (2000).</li>
<li>Lightfoot, J. T., Turner, M. J., Debate, K. A., Kleeberger, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interstrain+variation+in+murine+aerobic+capacity%5BTitle%5D)+AND+%22Med.+Sci.+Sports+Exerc%22%5BJournal%5D)">Interstrain variation in murine aerobic capacity.</a> Med. Sci. Sports Exerc. 33 (12), 2053-2057 (2001).</li>
<li>Turner, M. J., Kleeberger, S. R., Lightfoot, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+genetic+background+on+daily+running-wheel+activity+differs+with+aging%5BTitle%5D)+AND+%22Physiol.+Genomics%22%5BJournal%5D)">Influence of genetic background on daily running-wheel activity differs with aging.</a> Physiol. Genomics. 22 (1), 76-85 (2005).</li>
<li>Haight, T. J., van der Laan, M. J., Manini, T., Tager, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+effects+of+leisure-time+physical+activity+on+walking+speed%5BTitle%5D)+AND+%22J.+Nutr.+Health+Aging%22%5BJournal%5D)">Direct effects of leisure-time physical activity on walking speed.</a> J. Nutr. Health Aging. 17 (8), 666-673 (2013).</li>
<li>Studenski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gait+speed+and+survival+in+older+adults%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Gait speed and survival in older adults.</a> JAMA. 305 (1), 50-58 (2011).</li>
<li>Jacobsen, K. R., Kalliokoski, O., Teilmann, A. C., Hau, J., Abelson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+isoflurane+anaesthesia+and+vasectomy+on+circulating+corticosterone+and+ACTH+in+BALB%2Fc+mice%5BTitle%5D)+AND+%22Gen.+Comp.+Endocrinol%22%5BJournal%5D)">The effect of isoflurane anaesthesia and vasectomy on circulating corticosterone and ACTH in BALB/c mice.</a> Gen. Comp. Endocrinol. 179 (3), 406-413 (2012).</li>
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</ol>

The authors have no conflicts of interest to disclose.

Described here is a method to assess voluntary running endurance in laboratory mice using a motorized treadmill without the use of a shock grid. This method can reveal differences among sub-lines of C57BL/6 mice that differ in expression of the cytokine IL-15, which in turn causes differences in expression of factors that underlie exercise endurance16,17. In keeping with the principles of the &#8220;Three R&#8217;s&#8221; in laboratory animal science, this method can be used as a more humane alternative, or refinement, to forced exercise protocols that utilize a shock grid8-11. Forced exercise can also introduce the confounding effects of psychological stress and avoidance behavior12 on both running parameters and post-exercise measurements such as serum cytokines and hormones.
A limitation of the technique is that determination of sustained maximal running speed, refusal to run, and the end point of exhaustion are each assessed subjectively by the human observer. However, most young mice will run voluntarily if acclimated slowly to the apparatus; moreover, mice generally exhibit clear behavioral signs of refusal to run and of having reached exhaustion after running. Nevertheless, successful conduction of this technique requires some observer sensitivity to the animals. Therefore, it is recommended that a single observer, preferably treatment-blinded, be used for all of the run-to-exhaustion tests within a study to minimize observer bias in determination of these parameters. Despite the somewhat subjective nature of the endpoints for determination of running speed and time-to-exhaustion, this methodology is robust and can detect clear differences in running endurance due to upregulation16 or deletion17 of a single gene, IL-15.
This protocol was demonstrated with different sub-lines of C57BL/6 mice, and was not tested with other strains of laboratory mice. C57BL/6 mice were utilized because this is the most common genetic background utilized for transgenic and knockout mice18. A study by Lightfoot et. al.19 indicated that C57BL/6 mice performed in the low-medium range of exercise endurance among inbred strains of laboratory mice. However, that study utilized forced treadmill running with a shock grid and much higher running speeds, whereas in this technique, both the maximal sustained speed and the duration of running are individual to each mouse. Additionally, data solely from male mice was shown here. While the technique works with female mice (not shown), these are not generally utilized in studies of transgenic and knockout mice.
Since running in this protocol is voluntary, loss of data from some mice that refuse to run is unavoidable, and should be taken into account when estimating group sizes necessary for statistical significance. Additionally, representative data show that the tendency to refuse to run increases with age in wild-type C57BL/6 mice. Therefore this technique may not be suitable for determination of exercise endurance in very old mice. However, genetic manipulation to increase IL-15 expression significantly lowered the percentage of refusals in older mice, suggesting that refusal to run may also reflect decreased exercise capacity in older animals. Voluntary exercise frequency, as well as gait speed, decline with age in both rodents and humans, and can be reflective of declines in metabolic parameters and muscle mass 20-22. In this regard, the parameters of time-to-exhaustion and maximum sustained running speed were presented separately in the representative data. Distance run (m) can be calculated by multiplying time-to-exhaustion by maximum sustained running speed; however, this measure is somewhat inaccurate due to the initial phase of the run at slightly slower speeds. Some models of rodent treadmills have digital recording features that can be set to calculate distance run precisely; however, presenting time-to-exhaustion and speed separately may yield more information.
The reduced precision of endpoints, differences in running speed, and the complication of mice that refuse to run in the technique described here, are balanced by the consideration that the technique is more humane and less stressful to mice. However, blood markers of murine stress such as corticosterone levels were not determined to demonstrate rigorously that this technique is less stressful than forced exercise using a shock grid. Such determinations are technically difficult in acute exercise experiments because glucocorticoid levels are confounded by the anesthetics used in obtaining blood samples in mice23 and by exercise itself 24. One study25 indicated that chronic forced exercise using a shock grid induced both psychological and immune stress in mice, while voluntary wheel running (in which mice could control both the duration and speed of running) had the opposite effect. Close behavioral examination of the mice, combined with their demonstrated willingness to run and the long durations of voluntary running bouts, suggest mice are not unduly stressed by the protocol described here. Moreover, representative data indicate this protocol can reveal clear differences in running endurance due to introduction or deletion of a single gene product within an inbred strain 16,17. Therefore, this protocol represents a more humane and scientifically useful alternative to forced exercise for determination of exercise endurance in murine models.

Obesity, insulin resistance, and type 2 diabetes are interrelated metabolic disorders that exert profound effects on the health of both the U.S. and worldwide populations 1-4. Endurance exercise can prevent, as well as treat, these conditions5,6. Moreover, assessment of gait speed and endurance are utilized clinically as diagnostic tests for frailty, sarcopenia, and the consequences of other disorders such as chronic obstructive pulmonary disease, in human subjects 7.
The biochemical pathways underlying the beneficial effects of endurance exercise on body composition and insulin sensitivity are beginning to be elucidated, using genetically or pharmacologically modified mice that display enhanced or reduced exercise capacity 8-11. However, many such studies have utilized motorized treadmills equipped with shock grids to force mice to run 8-11. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. Such procedures may introduce psychological stress and avoidance behavior as confounding factors affecting experimental parameters12. Other methods of measuring endurance, such as quantitation of ambulatory activity using a beam-break apparatus or quantitation of in-cage wheel running, may be confounded by modulation of circadian cycles, anxiety, inadvertent training, or food seeking behavior12-16. Moreover, these procedures require single housing, another source of psychological stress for mice. Therefore, a direct measurement of exercise endurance not confounded by stress is needed.
To obviate these concerns, our laboratory has developed and validated a method to assess maximal running capacity and sustained running speed in untrained, na&#239;ve mice using a motorized treadmill not equipped with a shock grid. The space at the end of the running belt, where the shock grid usually resides, then becomes a platform for mice to rest, and provides a place for mice that refuse to run to sit until removed from the apparatus. Mice are encouraged to run by a human observer using gentle tapping or touching with a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. This method has been used to quantify differences in exercise endurance between genetically-modified and control C57BL/6 mice that differ in expression of the muscle derived cytokine interleukin-15 (IL-15)16,17. This method is a humane refinement that decreases the confounding effects of negative reinforcement caused by use of a shock grid.

<table><tbody><tr><td>Open Rodent Treadmill Exer-3/6</td><td>Columbus Instruments, Columbus OH</td><td>1050RM</td><td>This catalog number is for models without shock grid. Shock grids can be removed manually from older models (Eco 3/6)</td></tr><tr><td>Sani Cloth Germicidal Towlettes</td><td>PDI- Professional Disposables, Inc., Orangeburg, NY</td><td>usually ordered through local facility</td><td>Contains 10% isopropyl alcohol; any equivalent product can be used.</td></tr><tr><td>Tongue depressors</td><td>Any local supplier</td><td></td><td></td></tr><tr><td>Laboratory timer</td><td>Any supplier</td><td></td><td></td></tr><tr><td>IL-15 TG mice*</td><td>JAX, Bar Harbor, ME</td><td>011002</td><td>Murine IL-15 transgene expressed from modified human alpha-skeletal actin promotor with altered signal sequence to facilitate secretion.</td></tr><tr><td>C57BL/6J mice (wild-type)*</td><td>JAX, Bar Harbor, ME</td><td>000664</td><td>Control mice for IL-15 TG</td></tr><tr><td>IL-15 KO mice*</td><td>Taconic Farms, Germantown, NY</td><td>4269-M</td><td>Homozygous IL-15 &quot;knockout&quot; mice on C57BL/6 background</td></tr><tr><td>C57BL/6NTac (wild-type)*</td><td>Taconic Farms, Germantown, NY</td><td>B6-M</td><td>Control mice for IL-15 KO</td></tr><tr><td>*Optional; mouse lines and treatments can be specific to the experimental protocol.</td><td></td><td></td><td></td></tr></tbody></table>

assessment of murine exercise endurance without the use of a shock grid: an alternative to forced exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

This procedure for measuring exercise endurance accurately reflects the molecular and metabolic profile of different strains of C57BL/6 mice that differ in expression of the cytokine IL-15 16,17. Transgenic mice that overexpress IL-15 (IL-15 TG mice) exhibit significantly increased run-to-exhaustion times compared to littermate controls, while mice that lack IL-15 (IL-15 KO mice) exhibit significantly reduced run times prior to reaching exhaustion (Figure 1A). Published studies have shown that muscles are shifted towards a more oxidative phenotype in IL-15 TG mice16; conversely, muscles in IL-15 KO mice are shifted to a less oxidative phenotype17.These muscle phenotypes correlate with levels of spontaneous physical activity in a beam-break apparatus, and with exercise endurance using the voluntary run-to-exhaustion test. Wild-type control C57BL/6 mice from two different sources (in-house colony and a commercial supplier) do not exhibit significantly different run times (Figure 1A).
Maximal sustained running speed differed only in IL-15 KO mice (Figure 1B). Distance run (not shown) can also be calculated and analyzed statistically; however, separation of the two parameters (time-to-exhaustion and speed) yields more information.
An unavoidable limitation of this technique is that some mice refuse to run voluntarily. The percentage of young wild-type control mice that refuse to run is around 10% at 4 months of age. The percentage of refusals increases to 20% at 8 months of age, and increases to more than 60% by 16 months of age (Figure 2). However, the percentage of refusals in the older age group is significantly lower in IL-15 TG mice (Figure 2).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51846/51846fig1highres.jpg" width="500" /> 
Figure 1. Representative data from the voluntary run-to-exhaustion test. (A) Time to exhaustion; (B) Sustained running speed. For both panels, 4 strains of C57BL/6 male mice were tested: WT CON1 (wild-type littermate controls from in-house transgenic colony); IL-15 TG (interleukin-15 overexpressing transgenic mice16, littermates of WT CON1); WT CON2 (wild-type control mice, obtained commercially); IL-15 KO (interleukin-15 knockout mice17, obtained commercially). Bars represent mean + SEM; n = 7 - 29 mice per group. Data were analyzed by Kruskal-Wallis one-way ANOVA on ranks; bars with different superscripts are significantly different at P &#60;0.05. Data are reprinted from previous publications16,17, with permission from the journal.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51846/51846fig1highres.jpg" width="500" /> 
Figure 2. Percentage of mice refusing to run voluntarily increases with age. Male interleukin-15 overexpressing transgenic mice (IL-15 TG) and littermate controls, both on a C57BL/6 background, were tested in the voluntary run-to-exhaustion protocol at 4, 8, 12, and 16 months of age. Bars represent percentage of mice in each age group that refused to run (n = 11 - 29 mice per group). Data were analyzed by Fisher&#8217;s Exact Test; asterisk denotes a significant difference at P &#60;0.05 between control and IL-15 TG at 16 months of age. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51846/51846fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

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Nanyang Technological University

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19.1K Views.  VA Puget Sound Health Care System. A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Video: Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson&#8217;s disease.

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

Evaluation of Exercise Tolerance of Pigs Subjected to Exercise

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based therapeutic angiogenesis is a promising approach for treating patients with significant symptoms, despite optimal pharmacological therapy and invasive procedures. However, many promising cardiovascular gene therapy techniques have failed to accomplish expectations in clinical trials. One explanation is a mismatch between preclinical and clinical endpoints used to measure efficacy. In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of the capillary vessels calculated from histological sections. Apart from mortality and morbidity, endpoints in clinical trials are subjective, such as exercise tolerance and quality of life. However, the preclinical and clinical endpoints likely measure different aspects of the applied therapy. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches. In clinics, the main goal is always to alleviate patients' symptoms and improve their prognosis and quality of life. To achieve better predictive data from preclinical studies, endpoint measurements must be better matched to those in clinical studies. Here, we introduce a protocol for a clinically relevant treadmill exercise test in pigs. This study aims to: (1) provide a reliable exercise test in pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies, and (2) better match the endpoints between preclinical and clinical studies.

Author Spotlight: Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System  

The present protocol describes a large animal exercise test model to assess the functional capacity of the cardiovascular system for evaluating the efficiency of new therapies in the preclinical setting. It is comparable to a clinical exercise test.

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based ...

Genetics

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Medicine

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Biology

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. However, in certain cardiac diseases such as inherited arrhythmia syndromes, e.g., arrhythmogenic cardiomyopathy (ACM) or myocarditis, physical exercise may have negative effects on the heart leading to a proarrhythmogenic substrate production. Currently, the underlying molecular mechanisms of exercise-related proarrhythmogenic remodeling are largely unknown, thus it remains unclear which frequency, duration, and intensity of exercise can be considered safe in the context of disease(s).
The proposed method allows to study proarrhythmic/antiarrhythmic effects of physical exercise by combining treadmill training with real-time monitoring of the ECG. Implantable telemetry devices are used to continuously record the ECG of freely moving mice over a period of up to 3 months both at rest and during treadmill training. Data acquisition software with its analysis modules is used to analyze basic ECG parameters such as heart rate, P wave duration, PR interval, QRS interval, or QT duration at rest, during and after training. Furthermore, heart rate variability (HRV) parameters and occurrence of arrhythmias are evaluated. In brief, this manuscript describes a step-by-step approach to experimentally explore exercise induced effects on cardiac electrophysiology, including potential proarrhythmogenic remodeling in mouse models.

Electrocardiogram (ECG) is the key variable to understanding cardiac electrophysiology. Physical exercise has beneficial effects but may also be harmful in the context of cardiovascular diseases. This manuscript provides a method of recording real-time ECG during exercise, which can serve to investigate its effects on cardiac electrophysiology in mice.

Regular physical exercise is a major contributor to cardiovascular health, influencing various metabolic as well as electrophysiological processes. ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Podsumowanie

Przeglądaj więcej filmów

Assessing Functional Performance in the Mdx Mouse Model

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Real-Time Electrocardiogram Monitoring During Treadmill Training in Mice