The overall goal of this procedure is to determine voluntary exercise endurance. In laboratory mice, this is accomplished by first allowing the mouse to acclimate to the procedure room in its home cage for one to two hours. The mouse is then allowed to acclimate to the treadmill and the sound of the motor.
Next, the treadmill speed is gradually increased until the mouse runs comfortably at its maximal sustained rate. The final step is to determine when the mouse has reached exhaustion. Ultimately, the amount of time the mouse has run to exhaustion reveals a measure of exercise endurance without the confounding effects of stress from use of a shock grid.
The main advantage of this technique is that it is a refinement of existing methods for determining exercise endurance and mice, primarily because it does not use a shot grid to encourage the mice to run, thus reducing the confounding effects of stress, both for the animal and for the experiment. Decide in advance on experimental groups and post exercise procedures for mice. Place the treadmill on a sturdy cart or table in a quiet room dedicated to this procedure.
During the running trials, set the treadmill for mouse sized lanes and at an inclination appropriate to the protocol. The inclination of positive five degrees is used here to obviate muscle damage due to eccentric exercise that occurs during downhill running. Place an absorbent pad below the treadmill bell to catch feces.
Allow the mouse to acclimate to the procedure room in its transported home cage with cage mates for one to two hours. Select a single mouse and record its tag number. Also weigh the mouse and record its weight.
Place the mouse in the treadmill without the motor turned on to acclimate for about five minutes. Turn the power on without the treadmill belt running and let the mouse acclimate to the machine noise for another five minutes. Remove mice in the sedentary groups from the treadmill at this point and return them to their cage or conduct post exercise procedures as per the experimental design.
After acclimation, turn the belt to a low speed. When the animal is exploring the belt and not on the platform, 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. Start a laboratory timer Once the mouse is running at the initial speed of 10 meters per minute, turn the speed up slowly.
When the mouse is at the top of the belt at increments of one meter per minute about every two minutes, turn the speed down to the previous setting. If the mouse repeatedly runs in short bursts and not at a steady pace, most mice will run readily, but they sometimes stop for short periods. Use the tongue depressor to gently tap or lift the rear of the animal to encourage resumption of running.
Often just noticing the hand with the tongue depressor coming towards them causes the mice to run some more. Do not stop the timer for temporary interruptions in running. Determine the maximal sustained running speed of each mouse by adjusting the speed and watching the mouse record this speed.
Some mice will completely refuse to run. Such mice will run in short bursts. Then stop to groom or pedal that is sit on the platform and use their front legs on the belt.
The classic posture of a refusing mouse 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 five minutes of the trial whether the mouse will or will not run.
Remove and identify mice that refuse to run from the analysis, but do not count them as sedentary mice. Depending on your experimental design, mice that refuse can be retested on a different day. 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. Identify exhausted mice and stop the timer. Record the total amount of time spent running at all speeds.
Depending on the experimental design, either return the mouse to its cage or conduct immediate post-exercise procedures such as blood or tissue collection. As per institutional guidelines, 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 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. Exercise endurance accurately reflects the molecular and metabolic profile of different strains of C 57 black six mice that differ in expression of muscle derived cytokine IL 15 transgenic mice that overexpress IL 15 exhibit significantly increased run to exhaustion times compared to ate controls while mice that lack IL 15 exhibit significantly reduced run times prior to reaching exhaustion wild type controls. See 57 black six mice from two different sources do not exhibit significantly different runtimes.
Maximal sustained running speed differed only in IL 15 knockout mice. 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 four months of age, the percentage of refusals increases to 20%at eight months of age and increases to more than 60%by 16 months of age.
However, the percentage of refusals in the older age group is significantly lower in IL 15 transgenic mice. While attempting this procedure, it's important to remember to stay calm and relaxed and pay attention to the mouse's behavior. After watching this video, you should have a good understanding of how to use a treadmill to determine exercise endurance and mice without using e chakra.
