For strenuous running, we used a rodent treadmill machine (MK-680R5; ME Service, Tokyo, Japan), in which electrical shocks were applied to the grid behind the lane to stimulate the rats that failed to run spontaneously (Fig. 1a). Rats were forced to run with a 5% incline [5, 6]. Healthy rats were forced to run 30 km (20 m/min for 50 min, 5 days a week) over 6 weeks (Fig. 1b), and we examined the completion rate every week. Rats were observed during the running, and rats which stopped running in the grid over one minute despite electric stimulation were defined as drop out.Fig. 1Relationship between the number of rats that could continue to run and the distance rats run during strenuous running. a Treadmill for rats. b Protocol of strenuous running for 30 km for 6 weeks. c Survival rate of rats during the strenuous running