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

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.