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

Summary

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.

Abstract

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.

Introduction

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 patients^2,3), but many individuals still lack effective treatment. Furthermore, existing drug treatments for CRF have not been found to be broadly, if at all, efficacious^4-7.

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 performance^17-19. 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 capacity^21-25. 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 fatigue^28,29. 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.

Protocol

This procedure was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.

1. Preparation

1. 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.
2. Prior to training and testing mice, ensure that the treadmill is on a flat surface and set to the treadmill to desired angle of inclination (recommended angle of inclination: 10°, to be kept consistent throughout training and testing) and set the electric shock frequency and intensity appropriately (recommended: 2 Hz, 1.22 mA).
   NOTE: The electric shock used should produce no more than a mild tingling sensation when touched by an ungloved finger and should be delivered in a pulsatile fashion (with each shock lasting 200 msec).
3. Place a clean sheet of butcher's paper or an absorbent pad under the treadmill to collect fecal boli and urine during training and testing.
4. Place a sheet of paper or an absorbent pad over the third of the treadmill housing (i.e., the clear plastic lid that covers the treadmill lanes) furthest from the shock grid.
   NOTE: This step is optional, but will create a darker space and may provide additional encouragement to avoid the lower portion of the treadmill.
5. If planning to use a wire brush to provide additional motivation during training, ensure that one is readily available prior to beginning training sessions.
6. Ensure that any drug or method for inducing and/or alleviating fatigue is available and can be prepared or performed during Step 2.14.

2. Training Mice to Use the Treadmill

NOTE: Training is necessary to ensure that mice are familiar with the treadmill and task and can perform appropriately when tested. If the majority of mice being trained are receiving frequent shocks or otherwise performing poorly during any training session, additional training sessions should be performed. On the first day, most mice will be shocked several times. By the second day of training, mice should be rarely making contact with the grid. If a mouse displays consistently poor training performance, it should be removed from the study. For female C57BL/6NCr mice, this is a rare occurrence (less than 1% have been removed from studies due to poor training performance), but it should be noted that other strains may perform differently during training.

1. With the treadmill off (and speed set to 0 m/min), individually lift the mice by the tail and place mice into separate lanes of a mouse treadmill. Promptly turn on the corresponding grid after placing each mouse on the treadmill. Ensure that mice are placed directly on the treadmill belt.
   NOTE: The amount of time and distance each mouse is held by its tail should be minimized by placing the cage near the treadmill prior to transferring mice to the treadmill and/or allowing mice to stand on a solid platform (e.g., a wire cage lid) until they are near the treadmill and the experimenter is ready to place them in the treadmill.
2. Allow mice to freely explore the treadmill for 1-3 min or until each mouse has explored its lane and/or received at least one shock from the grid.
3. Turn on the treadmill and slowly increase the speed until it begins moving (approximately 1.5 to 3.0 m/min). Monitor all mice to ensure that they begin walking. If a mouse does not begin walking or walks toward the shock grid, be prepared to intervene by tapping the mouse with a wire brush or tail tickling.
4. Slowly increase the treadmill speed to 8 m/min. Start a timer and continue monitoring behavior.
5. Increase treadmill speed to 9 m/min at 5 min, 10 m/min at 7 min, and stop the treadmill at 10 min.
6. Allow the mice to briefly explore the treadmill, then remove and return each to its cage.
7. Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.
8. To train additional mice, repeat Steps 2.1 through 2.7.
   NOTE: Allow alcohol to dry prior to placing new mice on the treadmill.
9. On the second day of training, repeat Step 2.1. Turn on the treadmill and increase the speed to 10 m/min. Start a timer.
   NOTE: Treadmill speed can be increased more rapidly than on the first day of training.
10. Increase treadmill speed to 11 m/min at 5 min, 12 m/min at 10 min, and stop the treadmill at 15 min.
11. Remove mice and return them to their cages.
12. Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill. To train additional mice, repeat Steps 2.9 through 2.12.
13. Perform additional days (3 days) of training in the same manner as the second day.
    NOTE: This step is optional, but is strongly recommended if most or all mice (of the same sex and strain) being trained display difficulty with the task. Mice can generally perform well in Step 3 when they have been trained for 3 days (i.e., with one additional day of training), although additional or fewer days of training may be appropriate depending on their performance during the second training day and the duration of Step 2.14.
14. Allow at least one full day to pass in which the mice have no exposure to the treadmill before proceeding to Step 3.
    NOTE: Any drug(s) used to induce and/or alleviate fatigue should be administered during this step.
    NOTE: This time period can be varied in length and used to induce fatigue and/or test interventions to reduce or eliminate fatigue. If testing mice more than 7 days after completing training, a pilot study is recommended to verify that the mice used will perform during testing.

3. Treadmill Fatigue Test

NOTE: In this test, fatigue-like behavior is defined as spending 5 consecutive seconds in the "fatigue zone". The fatigue zone is defined as the region encompassing the portion of the treadmill belt within approximately 1 body length of the shock grid as well as the grid, itself. Prior to testing, ensure that the point delineating this zone is clear to the experimenter, such as by applying a mark to the top or side of the treadmill lanes.

1. Set the treadmill speed to 12 m/min. Do not start the treadmill. Ensure that shock grids are turned off.
2. Individually place mice into separate lanes of the treadmill. Turn on the corresponding grid immediately after placing each mouse on the treadmill.
3. Simultaneously start the treadmill and a stopwatch.
   NOTE: Do not intervene during testing except to remove mice that meet the criterion for removal (see Step 3.5).
4. Increase treadmill speed as indicated in Table 1. Carefully observe all mice throughout the test.
   NOTE: The treadmill speeds listed in Table 1 were selected based on observations from adult female C57BL/6NCr mice. Higher treadmill speeds may be appropriate for larger (e.g., outbred CD-1 mice) or more athletic mice.
5. If a mouse remains in the fatigue zone for 5 continuous sec, promptly remove the mouse from the treadmill and record the duration and distance it ran.
6. When no mice remain on the treadmill, stop the treadmill. Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.
7. To test additional mice, repeat Steps 3.1 through 3.6.
   NOTE: This step is optional.

Results

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 mice^10,13, was administered. All data presented are from...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Exer 3/6 Animal Treadmill	Columbus Instruments	1050-RM Exer-3/6
Stopwatch	Daigger	EF24490M
Wire brush	Fisher Scientific	03-572-5
Compressed air	Dust-Off	FALDSXLPW
Absorbent pads	Daigger	EF2175CX
Butcher paper	Newell Paper Company	4620510
Alcohol (70%)	Fisher Scientific	BP82011