To begin, prepare a tissue organ bath by inserting the oxytube needle valve into the water jacket tissue bath. Connect the oxytube to a gas cylinder and allow 20 PSI to fill the water jacket tissue bath. Prepare the surgery area by running an additional oxytube from the gas line to a crystallizing dish filled with Krebs-Henseleit light solution so the muscle can be fully submerged.
Next turn on all equipment including the dual mode muscle lever system, stimulator and signal interface. For EDL muscle dissection, first lay the mouse in either a right or left lateral recumbent position with the top hind limb stretched and toes touching the dissection board. Tent the skin with forceps and cut from the ankle joint to the hip area.
Once the muscle is exposed, cut around the ankle. Pull the skin up to expose the leg muscles clearly. Locate the fascia line that separates the tibialis anterior and gastrocnemius and separate them using dissection scissors to expose the knee tendons.
Then place the dissection scissors between the two exposed knee tendons. Blunt dissect a pocket while pulling the scissors away from the leg until the scissors reach the ankle to expose the EDL. Using a pre tied loop knot of a 4 0 silk surgical suture lace one end of the suture under the tendon closest to the knee.
Tie a double square knot above the proximal muscle tendon junction without placing it on the muscle or including the tendon. Cut above the knot. Gently pull the loop tied to the tendon and the EDL will emerge from the pocket.
Tape the loop to the dissection area to create tension in the EDL. Tie a double square knot using another pre tied loop knot at the distal muscle tendon junction without placing it on the muscle or including the tendon. Cut the tendon above the knot on the side closer to the leg to remove the whole EDL from the mouse.
Cut the extra suture away from the double square knots on the proximal and distal sides of the muscle and place the muscle in the aerated bath by the surgery area. Place the muscle on the servo motor lever rig by attaching the EDL vertically between suspended platinum electrodes. Attach the distal loop knot to the stationary hook and the proximal loop knot to the hook on the servo motor arm.
Then raise the tissue bath to submerge the muscle in the aerated Krebs-Henseleit solution. Before measuring the maximum isometric force of the EDL muscle, stimulate the muscle with a twitch to ensure the muscle has not been damaged during surgery. Then use the length knob on the muscle lever system to find a muscle length using twitch stimulation at appropriate tension.
Record the starting length of the muscle between the suture knots and volts and millimeters. Input measurements into the calibration portion of the program for starting length. Next, record the length of supra maximal tetanic force at L knot and measure the fibers from the suture knot to the suture knot again with calipers.
Find the sub maximal isometric force of EDL at L knot before and after the experiment to ensure fatigue did not occur from the stimulation protocol. Next, set up the necessary software to complete the avatar work lube technique using in vivo strain trajectories. Determine stimulation protocol based on strain trajectories and EMG activity from en vivo muscle.
Upload the scaled strain changes with scaled length excursion into the program. Input the starting length in volts and millimeters to calibrate results using pre-calculated stimulation onset and duration. Run the muscle through the scaled length changes with determined length excursion for two stimulation cycles.
Then save the data. Stimulate at optimal length or L knot using sub maximal activation to determine if fatigue has occurred. Then remove the muscle from the bath, cut loop knots from the muscle and dab the excess solution off the muscle.
Weigh the muscle and determine the physiological cross-sectional area using the standard formula. Plot force over time for the avatar and in vivo muscle and calculate the coefficient of determination R square by squaring the scaled correlation between target and avatar muscle force. In this study, mouse EDL was used as an avatar for rat medial gastrocnemius or mg.
Comparison of work loops of in vivo rat mg and ex vivo avatar experiments indicated that optimizing the stimulation protocol improves the fit to the in vivo rat MG force compared to EMG based activation. Moreover, high R squared between ex vivo force production of mouse EDL and in vivo force production of rat MG indicated good replication. An improved prediction of in vivo MG force using ex vivo mouse EDL across all locomotor speeds such as walking, trotting and galloping.
Also, the root mean square error decreased for all speeds after optimization.
