2.8. Muscle Mechanics

Grip strength was measured in mice using a Digital Grip Strength Meter (Bonther Equipments, Ribeirão Preto, Brazil). Three measurements were taken from each mouse, and the force was calculated as the average of the measurements and expressed in millinewtons (mN). This test was performed in the healthy and tumor-bearing mice groups studied in the AET protocol to verify its effectiveness in preventing the loss of muscle force induced by cancer cachexia.

Force measurements were performed as described previously [31,32]. Briefly, mice were anaesthetized with 2,2,2-Tribromoethanol (Sigma-Aldrich, Steinheim, Germany, 1 mL/100 g body weight, i.p.). An incision was made on the ankle to expose the TA muscle tendon, and a lateral incision on the thigh was made to expose the sciatic nerve, in which a platinum electrode was connected. The hind limbs were then fixed, and the TA tendon was connected to a force transducer (Biopac Systems, Goleta, CA, USA). Muscle force was recorded and analyzed using the AcqKnowledge System program, version 3.9.1.6 (Biopac Systems). Mice were submitted to external warming in order to maintain core temperature throughout the procedure. At the start of the experiment, the muscle was set to the optimum length (L0, defined as the length resulting in maximum twitch strength). Therefore, the force was determined by stepwise-increasing stimulation frequencies of 4, 20, 55, 75, 100, and 150 Hz, pausing for 30 s between stimuli to avoid effects due to fatigue [33]. Muscle force at 150 Hz was analyzed and absolutely expressed in mN, as well as normalized by the TA muscle mass (mN/g).