Exer 6m open treadmill

We used a motor-driven treadmill (Columbus Instruments, Exer-6M Open Treadmill) to assess running endurance. To encourage mice to run, tactile stimuli were provided by a manual light tap with a small paint brush to the hindquarters when the animal reached the back of the treadmill. On days 0 and 1, mice were placed on the treadmill to acclimate for 10 min with a speed of 10 m/min at 0% gradient. On day 2, running capacity was determined by placing mice on the treadmill without treadmill motion for 2 min for acclimation. Next, mice were subjected to a 5 min warm-up period at 10 m/min. Speed was then increased by 2 m/min each 2 min until a maximum of 20 m/min. Mice were then run to exhaustion, defined as sitting at the back wall for 10 s despite continual tapping with the brush. Work (Joules) was calculated from body weight (kg) × 9.8 × distance run in m.

Treadmill exercise tests were performed two weeks before the end of the study using a motorized rodent treadmill (Exer-6M Open Treadmill; Columbus Instruments, OH, USA). Before the exercise performance test, the mice were accustomed to the treadmill with a 5-min run at 7 m/min for 2 days. To evaluate the maximal aerobic speed, as previously described [35 (link)], the exercise test regimen was 10 m/min for the first minute, followed by 2 m/min increments every 2 min until 14 m/min, then 1 m/min every minute.

Atorvastatin treated mice received 10 mg/Kg/day via esophageal gavage [16] (link). The administered aqueous suspension was freshly prepared, in the concentration of 1 mg/ml, so that each mouse received 10 µl of suspension per gram of its weight. Exercised mice were gradually accustomed to the training program’s daily duration and speed over the first training week (full program: 5 times/week, 40 min/session, velocity = 12 m/min, slope = 5°), which was accomplished in a motorized treadmill (Exer-6 M Open Treadmill, Columbus Instruments, USA) with 6 separate compartments, so that an equal amount of animals could train simultaneously in an individual manner. Furthermore, stimuli on the treadmill were mostly mechanical, and electrical shock-plate was only used during the first session, and only in some individual mice, for them to get accustomed to the training procedure. During the exercise sessions, the mice were closely monitored for the verification of successful session completion. The animals that did not receive any of the two treatment modalities for the duration of the interventions, received the corresponding placebo treatment which included esophageal injection of 100 µl PBS, for non-atorvastatin receiving animals, and daily placement on the stable treadmill for 40 min for non-exercising animals.