We thank Dr. Nicolai Konow for providing the data used in this study. Funded by NSF IOS-2016049 and NSF DBI-2021832.

&quot;Avatar&quot;- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

<ol>
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All authors acknowledge there is no conflict of interest.

While organisms move seamlessly across landscapes, the underlying loads and strains that the muscles experience vary drastically1,6,23. During both in vivo locomotion1,24 and in &#34;avatar&#34; experiments, muscles are stimulated submaximally under cyclical, non-steady conditions. The isometric force-length and isotonic force-velocity relationships are not well...

Moving animals achieve impressive athletic feats of endurance, speed, and agility in complex environments. Animal locomotion is particularly impressive in contrast to human-engineered machines-the stability and agility of current-legged robots, prostheses, and exoskeletons remain poor compared to animals. Legged locomotion in natural terrain requires precise control and rapid adjustments to alter the speed and maneuver environmental features that act as unexpected perturbations1,2,3,4. Yet, understanding non-steady locomotion is inherently challenging because the dynamics depend on complex interactions between the physical environment, musculoskeletal mechanics, and sensorimotor control1,2. Legged locomotion requires responding to unexpected perturbations with rapid multi-modal processing of sensory information and coordinated actuation of limbs and joints1,5. Ultimately, movement is made possible by muscles producing force via intrinsic mechanical properties of the musculoskeletal system as well as from neural control1,5,6,7. An outstanding question of neuromechanics is how these factors interact to produce coordinated movement in response to unexpected perturbations. The following technique utilizes muscle&#39;s intrinsic mechanical response to deformation using in vivo strain trajectories during controllable ex vivo experiments with an &#34;avatar&#34; muscle.
The muscle work loop technique has provided an important framework for understanding intrinsic muscle mechanics during cyclical movements8,9,10. The traditional work loop technique drives muscles through predefined, typically sinusoidal, strain trajectories using frequencies and activation patterns measured during in vivo experiments2,8,9,11. Using sinusoidal length trajectories can realistically estimate work and power output during flight12 and swimming2 under conditions where animals do not undergo rapid changes in strain trajectories due to interaction with the environment and musculoskeletal kinematics. However, in vivo muscle strain trajectories during legged locomotion arise dynamically from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment5,7,13,14. A more realistic work loop technique is needed to emulate loads, strain trajectories, and force production that corresponds to in vivo muscle-tendon dynamics and provides insight into how intrinsic muscle mechanics and neural control interact to produce coordinated movement in the face of perturbations.
Here, we present a novel way to emulate in vivo muscle forces during treadmill locomotion by using an &#34;avatar&#34; muscle from a laboratory rodent during controlled ex vivo experiments with in vivo strain trajectories that represent time-varying in vivo loads. Using the measured in vivo strain trajectories from a target muscle on muscles from a laboratory animal during controlled ex vivo experiments will emulate loads experienced during locomotion. In the experiments described here, the ex vivo mouse extensor digitorum longus (EDL) muscle is used as an &#34;avatar&#34; for the in vivo rat medial gastrocnemius (MG) muscle during walking, trotting, and galloping on a treadmill13. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. While mouse EDL muscles differ in size, fiber type, and architecture compared to the rat MG, it is possible to control for these differences. The &#34;avatar&#34; technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

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			<td>Braided Non-Absorbable Silk Suture 4-0&#160;</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>S88 Stimulator</td>
			<td>Grass</td>
			<td>M643H05</td>
			<td>Available for purchase on Ebay</td>
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			<td>Sigma-Aldrich</td>
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			<td>Wild Type Mice</td>
			<td>Jackson Laboratory</td>
			<td>B6C3Fe a/a Ttn mdm/J</td>
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"avatar", a modified ex vivo work loop experiments using in vivo strain and activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics (&#39;preflexes&#39;) in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our &#34;avatar&#34; technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the &#34;avatar&#34; technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the &quot;Avatar&quot; Technique to Advance Muscle Mechanics Research 

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

The goal of the &#34;avatar&#34; experiments is to replicate in vivo force production and work output as closely as possible during ex vivo work loop experiments. This study chose to use mouse EDL as an &#34;avatar&#34; for rat MG because mouse EDL and rat MG are both comprised of mostly of fast-twitch muscles20,21. Both muscles are primary movers of the ankle joint (EDL ankle dorsiflexor, MG ankle plantarflexor) with similar pennation angles (m...

Watch this Scientific Journal Video about Bridging the Gap Between In Vivo and Ex Vivo Studies with the "Avatar" Technique to Advance Muscle Mechanics Research at JoVE.com

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and ...

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533 Views.  Northern Arizona University. This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response. 

Video: Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the "Avatar" Technique to Advance Muscle Mechanics Research 

"Avatar" Work Loop Technique Using In Vivo Strain Trajectories to Replicate Ex Vivo Muscle Force

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.