Model 305c

In vivo gastrocnemius force measurements were performed as described previously [24 (link)]. Mice were anesthetized and stainless steel-wired electrodes were placed on each side of the sciatic nerve. Torque production of the plantar flexors was measured using a muscle lever system (Model 305c; Aurora Scientific, Aurora ON, Canada). The force–frequency curves were determined by increasing the stimulation frequency in a step-wise manner, pausing for 30 s between stimuli to avoid effects due to fatigue. Following force measurements, animals were sacrificed by cervical dislocation and muscles were dissected and weighed. Force was normalized to the muscle mass as an estimate of specific force.

Skeletal muscle force in vivo was determined as described previously^{48 (link)}. Briefly, mice were anesthetized by intraperitoneal injection of xylazine (Xilor; 20 mg/kg) and Zoletil (10 mg/kg). Z small incision was made from the knee to the hip, to expose the sciatic nerve. Before the branch of the sciatic nerve, Teflon-coated 7 multi-stranded steel wires (AS 632; Cooner Sales, Chatsworth, CA, USA) were implanted with sutures on either side of the sciatic nerve. To avoid recruitment of the ankle dorsal flexors, the common peroneal nerve was cut. Torque production of the plantar flexors after nerve stimulation was measured using a muscle lever system (Model 305 C; Aurora Scientific, Aurora, ON, Canada). Normalized muscle force was determined by dividing absolute force by muscle mass.

In vivo functional testing of TA muscles was performed at 8-weeks post-injury. Briefly, in vivo physiological properties of the TA muscle were measured in anesthetized rats (isoflurane 1.5–2.0%) using a dual-mode muscle lever system (Model 305C, Aurora Scientific, Inc). Subcutaneous needle electrodes were inserted on either side of the common peroneal nerve, and an optimal voltage was set with a series of tetanic contractions (150 Hz, 0.1 ms pulse width, 400 ms train). Then, a skin incision was made at the antero-lateral aspect of the ankle and the distal tendons of the extensor digitorum longus (EDL) and extensor hallucis longus muscles were isolated and severed. TA muscle isometric tetanic torque was measured (10–200 Hz) with the ankle constrained at a right angle. This procedure was then repeated on the contralateral limb. For one animal in the bilateral VML injury, no treatment group, we were unable to measure torque in the contralateral limb because the TA tendon was accidentally severed. After all measurements were collected, the TA and EDL muscles were collected from each hindlimb, blotted, and weighed (wet).

A window was cut into the ventral thoracic cuticle, and overlying air sacs were removed. Contractions in the extensor tibiae muscle were elicited by stimulation of Nerve 3b (N3b) or Nerve 5 (N5) in the thorax, for SETi and FETi respectively, through a pair of 50 μm silver hook electrodes placed under the nerve and insulated with petroleum jelly. Stimulation strength was set just above the threshold for eliciting a single twitch reliably. The axon of common inhibitor motoneuron CI₁ runs in the same nerve as that of SETi, but it was not activated in these experiments. This was confirmed by making intracellular recordings from slow extensor tibiae muscle fibres during N3b stimulation. Such recordings revealed excitatory junctional potentials (from SETi) but no inhibitory potentials [62 ].
Stimuli were generated using a Master 8 stimulator (AMPI, Jerusalem, Israel). Series of pulses at various frequencies were delivered for 10 s or 1 s, for SETi and FETi respectively, with a 60 s gap between each stimulus train to allow the muscle to recover. FETi stimulus trains were restricted to 1 s to prevent damage to the apodeme insertion point. Muscle forces were measured using an isometric force transducer (Model 305C, Aurora Scientific, Canada), digitised at 5 kHz using a micro1401 interface and Spike 2 software (both Cambridge Electronic Design, Cambridge, UK).

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.