In Situ Muscle Force Measurement

In situ muscle force measurements were performed, as previously described [12 (link)]. Briefly, a nonsurvival surgery was performed to expose the left gastrocnemius muscle and calcaneal tendon, the latter of which was cut and attached via silk suture to the force lever arm and the leg stabilized by inserting a disposable monopolar needle (902-DMF37-S, Natus Neurology, WI, USA) through the knee joint. Twitch and tetanic force were recorded following stimulation of the sciatic nerve with 200 ms square pulses via insulated monopolar needles (F-E2M-48, Grass Technologies, RI, USA). A high-speed servo motor-based apparatus (Model 305C, Aurora Scientific, ON, Canada) was used to measure force output. Stimulation current and resting tension were adjusted to maximize twitch force produced by a single stimulus pulse. All subsequent isometric tetanic force data were collected at this stimulation current and resting tension. Isometric force frequency relationship was recorded after stimulation by a train of square wave stimuli and maximum isometric tetanic force was recorded. Animals were then euthanized.