Isometric Torque Assessment of Anterior Crural Muscles

In vivo isometric torque of the anterior crural muscles was assessed as previously described (Lowe et al., 1995 (link); Baumann et al., 2014 (link), 2020 (link)). The anesthetized mouse (see section Ethical Approval and Animal Models) was placed on a temperature-controlled platform to maintain core temperature at 37°C, and the left knee was clamped and the left foot was secured to an aluminum footplate that is attached to the shaft of the servomotor system (Model 300B-LR; Aurora Scientific, Aurora, Ontario, Canada). The proximal end of the nerve cuff, which was run subcutaneously to the dorsal cervical region, was then connected to a stimulator and stimulus isolation unit (Models S48 and SIU5, respectively; Grass Technologies, West Warwick, RI). For mice without nerve cuffs, sterilized platinum needle electrodes were precisely inserted through the skin for stimulation of the left common peroneal nerve and connected to the stimulator and stimulus isolation unit. The contractile function of the anterior crural muscles was assessed by measuring isometric torque as a function of stimulation frequency (torque frequency protocol; 20–300Hz; 150-ms train with 0.1-ms pulses). Peak isometric torque was recorded as the highest tetanic torque obtained during the torque-frequency protocol.