3 in 1 whole animal system

TA force measurements were performed using an Aurora Scientific 3-in-1 Whole Animal System, as previously described [38 (link),39 (link)]. Animals were anesthetized using 2.5% isoflurane and placed on the stage in the supine position. After adjusting the leg to the correct position and securing the foot of the animal against the footplate, the knee joint was stabilized to ensure isometric muscle contractions. Electrodes were placed subcutaneously above the TA muscle until the maximum twitch force was identified. The electrodes were then secured with clamps. Tetanic curves were generated following muscle stimulation at 150 Hz. Electrodes were adjusted as needed to achieve regularly shaped tetanic curves (an uninterrupted, sharp vertical increase at the start of stimulation, followed by a flat plateau, and then a sharp vertical decrease at the end of stimulation). A minimum of 4 tetanic contraction measurements were recorded per treatment group and the peak tetanic force was selected for downstream analysis. Force measurements of untreated VML and uninjured controls (n = 4) were recorded alongside those of the experimental groups.

The muscle contractility test was conducted using 3-in-1 Whole Animal System (Aurora Scientific) at 2 weeks after the ischemia injury. All instrumentations were turned on at least 30 min before testing for proper calibration and to minimize thermal drift of the force transducer. Mice were under general anesthesia with 2% isoflurane in oxygen inhalation and placed on a heated platform (37°C) during the whole testing process. An incision was made on the skin anterior to the ankle and the tendon of the tibialis anterior (TA) muscle was carefully isolated. A 4.0 Ethicon silk nonabsorbable suture was tied to the tendon. The other side of the suture was attached to a load cell as described previously.^{26 (link),27 (link)} After the optimal length of the muscle is achieved, the maximally fused tetanic contraction was obtained at 150 Hz at this length. The maximal specific force (mN)/muscle weight (mg) was used to evaluate muscle contractility.