This research was supported by the Intramural Research Program of the NIH, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Grant 1Z01 DK011006. We wish to thank Michele Allen for providing technical assistance, Eleni Solomos for editorial assistance, and the NIH veterinary and animal care staff for providing care for the mice used in developing this method.

This procedure was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.
1. Preparation
<ol>
	<li>To allow for rapid identification of each mouse prior to testing, tattoo the tails of all mice to be trained and tested with identifying marks. 
	NOTE: This step is optional. Permanent marker or other methods of identification can be used as an alternative to tattooing.</li>
	<li>Prior to training and testing mice, ensure that the tr...

<ol>
	<li>Hofman, M., Ryan, J. L., Figueroa-Moseley, C. D., Jean-Pierre, P., Morrow, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-related+fatigue:+the+scale+of+the+problem.">Cancer-related fatigue: the scale of the problem.</a> Oncologist. 12 Suppl 1, 4-10 (2007).</li><li>Schwartz, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+fatigue+patterns+and+effect+of+exercise+in+women+with+breast+cancer.">Daily fatigue patterns and effect of exercise in women with breast cancer.</a> Cancer Pract. 8 (1), 16-24 (2000).</li><li>Schwartz, A. L., Mori, M., Gao, R., Nail, L. M., King, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+reduces+daily+fatigue+in+women+with+breast+cancer+receiving+chemotherapy.">Exercise reduces daily fatigue in women with breast cancer receiving chemotherapy.</a> Med. Sci. Sports Exerc. 33 (5), 718-723 (2001).</li><li>Butler, J. 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=A+phase+III,+double-blind,+placebo-controlled+prospective+randomized+clinical+trial+of+d-threo-methylphenidate+HCl+in+brain+tumor+patients+receiving+radiation+therapy.">A phase III, double-blind, placebo-controlled prospective randomized clinical trial of d-threo-methylphenidate HCl in brain tumor patients receiving radiation therapy.</a> Int. J. Radiat. Oncol. Biol. Phys. 69 (5), 1496-1501 (2007).</li><li>Jean-Pierre, P., 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=A+phase+3+randomized,+placebo-controlled,+double-blind,+clinical+trial+of+the+effect+of+modafinil+on+cancer-related+fatigue+among+631+patients+receiving+chemotherapy:+a+University+of+Rochester+Cancer+Center+Community+Clinical+Oncology+Program+Research+base+study.">A phase 3 randomized, placebo-controlled, double-blind, clinical trial of the effect of modafinil on cancer-related fatigue among 631 patients receiving chemotherapy: a University of Rochester Cancer Center Community Clinical Oncology Program Research base study.</a> Cancer. 116 (14), 3513-3520 (2010).</li><li>Mar Fan, H. G., 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=A+randomised,+placebo-controlled,+double-blind+trial+of+the+effects+of+d-methylphenidate+on+fatigue+and+cognitive+dysfunction+in+women+undergoing+adjuvant+chemotherapy+for+breast+cancer.">A randomised, placebo-controlled, double-blind trial of the effects of d-methylphenidate on fatigue and cognitive dysfunction in women undergoing adjuvant chemotherapy for breast cancer.</a> Support. Care Cancer. 16 (6), 577-583 (2008).</li><li>Moraska, A. 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=Phase+III,+randomized,+double-blind,+placebo-controlled+study+of+long-acting+methylphenidate+for+cancer-related+fatigue:+North+Central+Cancer+Treatment+Group+NCCTG-N05C7+trial.">Phase III, randomized, double-blind, placebo-controlled study of long-acting methylphenidate for cancer-related fatigue: North Central Cancer Treatment Group NCCTG-N05C7 trial.</a> J. Clin. Oncol. 28 (23), 3673-3679 (2010).</li><li>Schwartz, A. L., Thompson, J. A., Masood, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-induced+fatigue+in+patients+with+melanoma:+a+pilot+study+of+exercise+and+methylphenidate.">Interferon-induced fatigue in patients with melanoma: a pilot study of exercise and methylphenidate.</a> Oncol. Nurs. 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T., 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=Strain+screen+and+haplotype+association+mapping+of+wheel+running+in+inbred+mouse+strains.">Strain screen and haplotype association mapping of wheel running in inbred mouse strains.</a> J. Appl. Physiol. 109 (3), 623-634 (2010).</li><li>Bartolomucci, A., 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=Individual+housing+induces+altered+immuno-endocrine+responses+to+psychological+stress+in+male+mice.">Individual housing induces altered immuno-endocrine responses to psychological stress in male mice.</a> Psychoneuroendocrinology. 28 (4), 540-558 (2003).</li><li>Martin, A. L., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lonely+mouse:+verification+of+a+separation-induced+model+of+depression+in+female+mice.">The lonely mouse: verification of a separation-induced model of depression in female mice.</a> Behav. Brain Res. 207 (1), 196-207 (2010).</li><li>V&#245;ikar, V., Polus, A., Vasar, E., Rauvala, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+individual+housing+in+C57BL/6J+and+DBA/2+mice:+assessment+of+behavioral+consequences.">Long-term individual housing in C57BL/6J and DBA/2 mice: assessment of behavioral consequences.</a> Genes Brain Behav. 4 (4), 240-252 (2005).</li><li>Salem, G. H., 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=SCORHE:+a+novel+and+practical+approach+to+video+monitoring+of+laboratory+mice+housed+in+vivarium+cage+racks.">SCORHE: a novel and practical approach to video monitoring of laboratory mice housed in vivarium cage racks.</a> Behav. Res. Methods. 47 (1), 235-250 (2015).</li><li>Courtney, S. M., Massett, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+exercise+capacity+QTL+using+association+mapping+in+inbred+mice.">Identification of exercise capacity QTL using association mapping in inbred mice.</a> Physiol. Genomics. 44 (19), 948-955 (2012).</li><li>J&#248;rgensen, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alpha-AMPK+knockout+on+exercise-induced+gene+activation+in+mouse+skeletal+muscle.">Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle.</a> FASEB J. 19 (9), 1146-1148 (2005).</li><li>Knab, A. M., Bowen, R. S., Moore-Harrison, T., Hamilton, A. T., Turner, M. J., Lightfoot, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability+of+exercise+behaviors+in+mice.">Repeatability of exercise behaviors in mice.</a> Physiol. Behav. 98 (4), 433-440 (2009).</li><li>Lightfoot, J. T., Turner, M. J., Debate, K. A., Kleeberger, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+variation+in+murine+aerobic+capacity.">Interstrain variation in murine aerobic capacity.</a> Med. Sci. Sports Exerc. 33 (12), 2053-2057 (2001).</li><li>Lightfoot, J. T., 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=Quantitative+trait+loci+associated+with+maximal+exercise+endurance+in+mice.">Quantitative trait loci associated with maximal exercise endurance in mice.</a> J. Appl. 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The current protocol describes how to use a mouse treadmill to measure fatigue-like behavior. This method has several advantages over VWRA, a common preclinical assay of fatigue-like behavior. VWRA requires that mice choose to interact with the test apparatus. As a result, some inbred strains of mice rarely interact with the wheel16 and run so little that it may be difficult or impossible to identify a fatigue-induced decrease in activity. In contrast, the treadmill fatigue test eliminates that choice and ther...

Fatigue affects a wide range of people, can markedly reduce quality of life, and frequently has an unclear or unknown pathogenesis. Cancer-related fatigue (CRF), for example, is experienced by the majority of cancer patients undergoing treatment and can persist long after cancer treatment has been completed and in the absence of detectable cancer1. Moreover, fatigue is also a prominent symptom in numerous other diseases and disorders, including chronic fatigue syndrome, depression, diabetes, and fibromyalgia. Fortunately, there are non-pharmacological interventions that are capable of helping some people experiencing fatigue (e.g., exercise can reduce CRF for some breast cancer patients2,3), but many individuals still lack effective treatment. Furthermore, existing drug treatments for CRF have not been found to be broadly, if at all, efficacious4-7.
Despite the therapeutic need and lack of drug treatment options, preclinical assays of fatigue to aid in the discovery and development of novel fatigue treatments are lacking, especially in animal models. One of the only preclinical assays of fatigue for rodent studies is voluntary wheel running activity (VWRA)9-15, in which mice or other rodents are given free access to a running wheel and their daily running activity is recorded. In many studies, VWRA is the only measure of fatigue-like behavior, with fatigue-like behavior defined (in either VWRA or the current protocol) as a decrease in the measured physical activity in the experimental group. Although VWRA can provide a useful longitudinal measure of fatigue-like behavior, it is a relatively low-throughput assay, running varies considerably between inbred mouse strains16, and it requires subjects to be individually housed, which may cause changes in behavior and test performance17-19. Other assays, such as home cage behavioral monitoring and analysis, can also provide continuous data collection and some systems may allow for subjects to be housed in pairs20. These assays have utility, but may be less sensitive as a means of detecting fatigue-like behavior and, like wheel running, are also low-throughput.
In contrast to VWRA, mouse treadmill tests do not rely upon voluntary activity and can be completed in a short time frame, allowing for higher throughput. In comparison to VWRA, these tests employ external motivators. Specifically, there is usually an electrified metal grid located to the rear of the moving belt to provide mice with an electric shock should they cease to run. In addition to this shock grid, mice may be motivated to run on the treadmill via several other methods, including prodding, poking, or touching them with a hand, brush, or other tool and directing short puffs of air at them. Instead of fatigue, mouse treadmill tests are often used to measure aerobic and/or anaerobic exercise capacity21-25. Mice are motivated to run until they are incapable of or unwilling to continue running on the treadmill as a means of escaping further electric shocks. Testing then ends when mice meet the criterion for exhaustion. In these protocols, to ensure that mice reach true physiologic exhaustion, the criterion for exhaustion is often defined as spending five continuous seconds laying on top of the shock grid and failing to continue running in the face of repeated aversive stimuli. Thus, fatigue-like behavior may be masked in typical treadmill tests due to the strong aversive nature of the external motivation and criterion for ending the test. Interestingly, and in contrast to many other studies using rodent treadmills, a recent publication describes another version of a treadmill fatigue test, which was used as part of an examination of the effects of social stress in mice26. Although the method used by this group markedly differed from the current protocol (i.e., they employed a single-lane treadmill and required 10 sec of electric shock as the criterion for ending their test), their study highlights the utility of and interest in developing a quick, simple fatigue test using the mouse treadmill.
Fatigue is likely to be detectable by means other than wheel running and alterations in routine behaviors. CRF makes patients feel exhausted by a lesser amount of muscle fatigue, as determined by electromyographic analysis, than people without CRF27. Additionally, reduced motivation has been noted in and is measured by several scales measuring human fatigue28,29. Thus, a useful preclinical assay of fatigue-like behavior should distinguish between healthy and fatigued mice on the basis of a measure other than physiological capability and should not obscure decreases in motivation. To achieve that end while avoiding limitations of VWRA and other assays, the current method was developed by adapting the mouse treadmill test. This method uses a shock grid as the sole external motivator to make mice run on the treadmill. Mice quickly learn that the grid provides an aversive stimulus and will promptly move away from it when placed on the treadmill and maintain some distance from it when running.
When mice fatigue, they spend progressively more time toward the back of the treadmill instead of maintaining speed toward the front end. Therefore, the criterion for test completion in this protocol is spending five continuous seconds in the designated fatigue zone (i.e., the rear of the treadmill, ranging from approximately one body length from the shock grid to, and including, the shock grid). This takes advantage of the aversive nature of the grid without requiring mice to receive many or any actual shocks after training. By allowing mice to complete testing using the current criterion rather than exhaustion (as defined above), this method provides a means of using the treadmill to measure fatigue-like behavior rather than its maximal (or near-maximal) physiological capability. Thus, this method can provide a simple, high-throughput assay of fatigue-like behavior in mice and can serve either as an independent or complementary measure to other assays of fatigue-like behavior.

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			<td height="21" style="height:21px;width:264px;">Exer 3/6 Animal Treadmill</td>
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			<td>Fisher Scientific</td>
			<td>03-572-5</td>
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			<td height="21" style="height:21px;">Compressed air</td>
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			<td height="21" style="height:21px;">Absorbent pads</td>
			<td>Daigger</td>
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			<td height="21" style="height:21px;">Butcher paper</td>
			<td>Newell Paper Company</td>
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			<td height="21" style="height:21px;">Alcohol (70%)</td>
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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.

This protocol allows fatigue-like behavior to be measured in mice using a treadmill. The data presented in this section was obtained by training and testing 3 separate groups of mice using the current protocol (excluding Figure 1A and 1C). To induce fatigue, 5-fluorouracil (5-FU), a cytotoxic chemotherapy drug known to cause fatigue in humans30 and fatigue-like behavior in mice10,13, was administered.&#160;All data presented are from...

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

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 ...

The overall goal of this procedure is to measure fatigue-like behavior in mice to establish a fatigue model, and test whether intervention can reverse the fatigue. This method can help answer key questions relating to fatigue, such as what interventions can help reduce fatigue or reverse fatigue models. The main advantage of this technique is that it can quickly assess fatigue-like behavior and or anti-fatigue interventions with a single piece of hardware.Visual demonstration of this method is critical. It is important to see and understand how the mice behave in order to successfully perform this task. Prior to training and testing mice, ensure that the treadmill is on a flat surface, and set it to the desired angle of inclination, which is usually 10 degrees.Next, ensure that the electric shock frequency and intensity are set appropriately, which is usually to two hertz and 1.22 milliamps. Under the treadmill, place a piece of paper or an absorbent pad to collect feces and urine. Then, place a pad over one third of the treadmill housing furthest from the shock grid to create a darkened space and entice the mice to stay in that area.Now, proceed with training the mice. With the treadmill off and the speed set to zero, quickly load the mice, holding them by their tails, directly onto the treadmill belt. After placing each mouse, promptly turn on its corresponding shock grid.First, allow the mice to explore freely. Stop the exploring time once each mouse has received at least one shock, or after about three minutes. Then, turn on the treadmill and slowly increase the speed until it begins moving at about 1.5 to 3.0 meters per minute.Ensure that all the mice begin walking. If a mouse does not begin walking, or walks toward the shock grid, tap the mouse with the wire brush or use tail tickling to encourage it to walk on the treadmill. Now, with all the mice walking, slowly increase the treadmill speed to eight meters per minute.Try to ensure that mice are walking forward on the treadmill while increasing the speed during this first training session. If mice are exploring are turning to face the shock grid, cease increasing treadmill speed until they resume walking. Then start a timer and continue monitoring the behavior.After five minutes, increase the speed to nine meters per minute. After seven minutes, increase the speed to 10 meters per minute. After 10 minutes, end the training session by stopping the treadmill.Allow the mice to briefly explore the treadmill a little longer, and return them to their home cage. It is not necessary to have all mice from one cage running at the same time, but try to minimize the number of times mice are removed from any given cage. Between sessions, clean the treadmill and shock grids with alcohol wipes.Also, replace the paper or absorbent pad beneath the treadmill. On the second day of training, repeat the process used on the first day, but increase the treadmill speed to 10 meters per minute before starting the timer. After five minutes, increase the treadmill speed to 11 meters per minute.After 10 minutes, increase the speed to 12 meters per minute and let the treadmill run until 15 minutes have expired. Then, end the training run. Additional days of training can be performed if the mice are having trouble with the task.Once trained, allow at least one full day of rest before the fatigue test. A gap of up to seven days is possible without retraining. In this test, fatigue-like behavior is defined as spending five consecutive seconds in the fatigue zone.The proper technique for removing mice involves gripping near the base of the tail and promptly lifting them out of the treadmill. The fatigue zone is the region of the treadmill belt within a body length of the shock grid, as well as the grid itself. Prior to testing, apply marks to the top or side of the treadmill lanes to delineate the fatigue zone.Next, set the treadmill speed to 12 meters per minute, but do not start the treadmill. Also, make certain that the shock grids are turned off. Now, individually place the mice into their separate lanes and immediately turn on the corresponding shock grid after each mouse is loaded.To begin the test, simultaneously start the treadmill and a stopwatch. Increase the treadmill speed as indicated. This schedule is based on testing female C57 Black 6 mice and may need to be modified to accommodate differently abled mice.Carefully observe all mice throughout the test, but do not intervene to help or motivate mice that enter the fatigue zone. Do not use the wire brush or the tail tickling techniques. Only intervene when a mouse remains in the fatigue zone for five continuous seconds.When this occurs, promptly remove the mouse from the treadmill and record the duration and distance it ran. It is critical that the experimenter carefully observes all the mice on the treadmill, and quickly removes those that have spent five continuous seconds in the fatigue zone. Once no mice remain on the treadmill, prepare for the next run.Mice were trained for five days, and then, to induce fatigue, 5-fluorouracil was administered for five days. This drug is known to cause fatigue in humans and fatigue-like behavior in mice. In the exercise capacity test, which uses tail tickling and air puffs to motivate mice during testing, no difference was seen between groups.On the following day, however, the mice were tested using the treadmill fatigue test. This test revealed a significant difference in the distance run during testing between chemotherapy treated and control mice. To validate that the treadmill fatigue test was measuring fatigue-like behavior, mouse voluntary wheel running was measured in a separate experiment during the five days of 5-fluorouracil treatment, and for an additional night beyond the final day of treatment.Similar results were obtained. In a different experiment, the treadmill fatigue test was also able to detect fatigue-like behavior in mice that had been treated with a different dose of 5-fluorouracil, once weekly, for two weeks. When untreated mice were tested regularly, they became less willing to run upon repeated testing.This data suggests that testing with this protocol should be limited to a single test to avoid a high rate of non-compliant mice. While attempting this procedure, it is important to observe your mice closely during the training and testing. Do not intervene until the criterion for fatigue has been met.After watching this video, you should have a good understanding of how to measure fatigue-like behavior in mice using this protocol, how mice behave on the treadmill, and how to quickly remove mice from the treadmill.

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Research

JoVE Journal

Behavior

18.1K vues.  National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. The overall goal of this procedure is to measure fatigue-like behavior in mice to establish a fatigue model, and test whether intervention can reverse the fatigue. This method can help answer key questions relating to fatigue, such as what interventions can help reduce fatigue or reverse fatigue models. The main advantage of this technique is that it can quickly assess fatigue-like behavior and or anti-fatigue interventions with a single piece of hardware.Visual demonstration of this m...