2.2. Training Procedures, Forelimb Grip Strength Test, and Maximal Workload

All mice in the training group performed 1 h of forced treadmill running every training day. Training was done 5 days/week. The running speed was set to 22.5 m/min. The treadmill (Natsume, Tokyo, Japan) was set to a five-grade incline. The training period lasted 8 weeks.

A grip-strength meter (GPM-100; Melquest, Toyama, Japan) was used to measure forelimb grip strength. As a mouse grasped the bar, the peak pull force in grams was recorded on a digital force transducer. In the conventional version of this test, the mouse is allowed to grasp the bar mounted on the force gauge. The gauge is reset to 0 g after stabilization, and the tail of the mouse is slowly pulled back by the inspector. Tension is recorded based on the gauge at the time the mouse released its forepaws from the bar. For the modified version of this test used in the current study, the gauge was rotated vertically and fixed to the metal stand to keep the system immobilized. The measurement procedure was identical to that in the conventional test, except for the direction in which the tail of the mouse was pulled by the inspector. For each test, trials in which only one forepaw or the hindlimbs were used, as well as those in which the mouse turned during the pull or left the bar without resistance, were excluded. Given that the speed of the tail pull can influence the measurement, we conducted the procedure at a constant speed that was sufficiently slow to permit mice to build up resistance against it. We performed three consecutive measurements per test at 30-second intervals.

Maximal exercise capacity was evaluated using a treadmill, according to a previously described method [20,21]. One week before training began, all mice were familiarized with running on a motorized rodent treadmill, as previously described [14]. After familiarization, mice performed two graded exercise performance tests at two time points, the first at 3 days before training, and the second 3 days after the 8-week training period. Tests started at 9 m/min for 9 min, going up to 10 m/min, and increasing by 2.5 m/min every 3 min. The starting incline was 0 and was raised by 5 every 9 min, with a maximal incline of 15. Exhaustion was defined as the inability of mice to continue regular treadmill running despite stimulation by repeated tapping on the back. At this point, running time (in min) was recorded, and each mouse was removed from the treadmill and returned to its home cage. Exercise capacity was expressed in time (min) and work (kg·m). The work performed (kg·m) was calculated as the product of body weight (kg) and vertical distance (m), where:

where θ was the angle of the treadmill, from 0 to 15 [15,16]. A second pair of exercise performance tests was completed after the training period, and changes in exercise capacity were calculated.