Exer 3 6 treadmill

This exercise regimen was modified from a Treat-NMD protocol (http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1.001.pdf). The Exer 3/6 treadmill (Columbus Instruments, USA) was used for the exercised cohorts. Exercise commenced at 12 weeks of age and mice were exercised 3 times every 2 weeks for a 12 week period. Mice were allowed 2 minutes for familiarisation. The exercise regimen was started at 5 m/min and gradually increased in 1 m/min increments to 10 m/min for the first 2 exercise days. For the following 6 exercise sessions, mice were run for 45 minutes with the speed of the treadmill incrementally increased to 12 m/min. The mice were run at 12 m/min for 45 minutes for the remaining exercise sessions.

Mice (N = 9 per group) were trained to run on an Exer 3/6 treadmill (Columbus Instruments, Columbus, OH) during four separate sessions spanning over 2 weeks preoperatively. For training sessions, mice walked on the treadmill with a 10° incline at a speed of 10 m/min for 10 min and then 15 m/min for 5 min with shocks enabled at 1 Hz. After the training period, mice were randomly separated into two groups. For treadmill exhaustion testing, mice were placed on the treadmill and allowed to acclimate with the belt off and shocks on for 5 min. The treadmill was then started at a speed of 5 m/min at a 10° incline and speed was ramped up by 1 m/min². Distance recording started when speed reached 10 m/min, after which speed was further increased to 15 m/min after 5 min, and then incrementally increased by 3 m/min every 5 min thereafter until maximum speed of 30 m/min. Exhaustion was defined as 40 shocks, at which point shocks were disabled and total walking distance was recorded. Treadmill exhaustion testing was performed on POD 7, 10, 14, and 21.

Mice were familiarised to motorised rodent treadmills (Exer 3/6 treadmill, Columbus Instruments) daily for 5 days prior to the commencement of the exercise protocol by walking for 15–20 min at a belt speed of 4–6 m/min. The exercise protocol consisted of nine consecutive days of running for 1 h/day (belt speed 8–14 m/min, at zero inclination). Gentle hand prodding was used during the training phase to motivate animals to run. SED mice were placed on stationary treadmills for the same duration, to control for the effects of handling and exposure to a new environment.

Exercise capacity was assessed using an exhaustion incremental treadmill test. This procedure was previously described in detail [11 (link)]. Briefly, each animal was placed on the belt of a 6-lane motorized treadmill (Exer 3/6 Treadmill, Columbus Instruments, OH, USA), supplied with shocker plates, which could be individually enabled or disabled for each lane. During each exhaustion test the electrical stimulus was fixed at 200 ms duration, 0.34 mA amplitude, and 1 Hz repetition rate.
After acclimatization, all mice were subjected to initial exhaustion treadmill tests at 0° inclination according to the following protocol: 5 min at 5 m/min and followed by incremental increase of speed of 1 m/min every min until exhaustion. Exhaustion was defined as spending time on the shocker plate without attempting to reengage the treadmill within 20 s. Three tests were performed on each animal, allowing 4 days between each test. Values obtained were averaged, providing a single value per animal. In a second set of experiments, endurance was evaluated as the time spent on the treadmill belt running at 50% of the maximal velocity reached in the previous exhaustion incremental test.

The experimental procedures reported below include two kinds of tasks: a T-maze decision task (effort-based choice) and a run (fatigue) procedure. In the latter, the run velocity of each animal was specific to its own aerobic capacity. Thus, before all the experimental procedures, mice were submitted to incremental exercise testing on a motor treadmill.
The multiple-lane treadmill (Exer-3/6 treadmill, Columbus Instruments, Ohaio, USA) was placed in the climatic chamber for training. Stainless steel grids at the end of the lines provided an electrical stimulus of 0.25 mA, 1 Hz, and 200 ms length, to keep the mice running, and brushes prevented the mice from pinching feet between grid and treadmill. The intensity of exercise was increased by 3 meter/min (6–33 meter/min) every 3 min at 0% grade until exhaustion, which was defined as the point at which mice touched the end of treadmill five times in one minute. This test provided the total distance run and the peak workload (PW) for each animal, with the aim to determine aerobic capacity and exercise training intensity for each animal. Based on the latter, individual workloads corresponding to 40%, 60% and 80% peak workload (used in Experiments 1–3 below) were determined for each animal. Total distance run varies in a range between 385 to 411 m (mean = 394) and PW in a range between 18 to 21 m/min (mean = 20).