1300 a whole mouse test system

After 8 weeks of treatment in situ assessment of the TA was performed using the 1300A Whole Mouse Test System and 701C stimulator (Aurora Scientific). The mice were anaesthetized using isofluorane inhalation and placed on a heated platform (37°C) for the procedure. Briefly, a small incision was made in the distal end of the animal’s leg and the skin is retracted halfway up the leg to expose the TA. The distal tendon of the TA was surgically isolated and the knee joint exposed. Surgical silk was used to secure the tendon to the dual-mode lever arm and the foot and knee were secured. The muscle was then stimulated to contract by placing electrodes adjacent to the sciatic nerve. The optimal length (Lo) was determined based on production of maximum twitch force (Pt), resting muscle length was recorded. The TA was then stimulated to contract (5 – 200Hz, with 2 minutes rest between each contraction) to generate a force frequency curve and maximal tetanic force (Po) was achieved at 150Hz. Absolute force (mN), specific force (mN/mm²), and the rate of muscle fatigue and recovery (%) were determined as previously outlined in Garton, et al. 2018 [17 (link)]. An additional control group of standard chow fed C57/BL6 mice (n = 10) were compared to the Nf1_Prx1^-/- test groups. The operator was blinded to treatment in all cases.

To measure the force independent of innervation, we isolated the TA in a bath of oxygenated Ringer’s solution and stimulated it with plate electrodes. Immediately after euthanasia, the distal tendon of the TA, the TA, and the knee (proximal tibia, distal femur, patella, and associated soft tissues) were dissected out and placed in Ringer’s solution (Sigma) maintained at 25 °C with bubbling oxygen with 5% carbon dioxide. The proximal tibia was sutured to a rigid wire attached to the force transducer and the distal tendon was sutured to a rigid fixture. No suture loops or slack was present in the system. The contralateral limb was immediately dissected and kept under low passive tension in oxygenated Ringer’s solution bath until measurement. Supramaximal stimulation voltage was found and the active force-length curve was measured in a manner similar to the in vivo condition. After measurement, the muscle was dissected free and the mass measured. An Aurora Scientific 1300-A Whole Mouse Test System was used to gather force production data.