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

"Avatar"- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

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</ol>

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 suited for predicting muscle force under these conditions2. Understanding the effects of non-steady strain (i.e., transients) and loading is essential for predicting force production during in vivo movement, and therefore is the main rationale for developing these &#34;avatar&#34; experiments2. &#34;Avatar&#34; experiments allow us to control muscle loading and strain trajectories while measuring force output. The &#34;avatar&#34; technique investigates the force response of muscles under in vivo-like conditions, without confounding factors of neural control and tendon compliance. To perform the &#34;avatar&#34; experiments, researchers will need a program that allows a muscle to go through prescribed length changes with the ability to stimulate at different starting lengths and for varying durations (see Supplementary Figure 3 for the program the authors use). Researchers need to specify starting muscle length (mm), length of excursion (mm), onset of stimulation (% of cycle duration) and duration of stimulation (ms) before doing experiments (see steps 1.3-1.4to obtain values for these parameters). In general, it is often desirable to select strides that are representative of all strides in the trial (e.g., start and end at a similar length, reach a similar peak force, have average EMG activity, etc.). Determining whether EMG/activation and force data from a selected stride is representative of other strides in the same trial can be helpful for &#34;tuning&#34; later, which can be done by plotting work loops (force versus length) of the entire trial using the target animal&#39;s muscle. During bipedal and quadrupedal locomotion, the shortest length to shortest length generally demarcates an entire stride (toe-off to toe-off), but EMG activation can vary. In some animals and muscles, EMG activation is closely correlated to foot contact, such as the rat MG shown here22. In other animals, such as the guinea fowl lateral gastrocnemius, EMG activation generally occurs at the longest length to achieve more stability during unknown terrain25. 
To perform &#34;avatar&#34; experiments, it is important to minimize the noise in the ex vivo force data. Force measurements are sensitive to several issues, including but not limited to tearing of the muscles during surgery, compliance of the sutures if the loop-knots are too long, improper scaling of the length inputs and excursion, and muscle fatigue. Tearing of the muscles often occurs when &#34;dissecting the pocket&#34; (step 2.2.3) and tying the loop-knot around the proximal portion of the tendon (step 2.2.4). While &#34;dissecting the pocket&#34;, keeping the dissection scissors flat and horizontal to the muscle will prevent the tips from nicking the EDL. Additionally, pulling the dissection scissors away and distally while blunt dissecting will also limit contact between the dissection scissors and the EDL muscles. Additionally, muscles should be kept damp with Krebs-Henseliet solution during the surgery preparation and when being used on the rig.
Properly scaling length inputs is more complicated. Muscle passive and active force can be affected if the starting length and/or excursion are not scaled properly. The ex vivo rise in passive tension should match the in vivo rise in passive tension (see Figure 1). One scaling issue that has been observed in previous experiments is that both passive and active tension can be affected if the length excursion (starting length to longest length) is too small or too large. Theoretically, muscles should reach peak force near their optimal length (L0)26, which is why we use optimal length (L0) to scale in vivo muscle lengths in ex vivo &#34;avatar&#34; experiments to accurately replicate in vivo force production. Architectural differences between muscles will play a role in determining the starting length and length excursion parameters. Although optimal length (L0) is found during supramaximally stimulated isotonic and isometric conditions, using it as a scaling metric in &#34;avatar&#34; experiments can potentially highlight limitations of the force-length and force-velocity relationships during cyclical movement that need more investigation. In most steady-state conditions, muscle&#39;s instantaneous length, velocity, and activation (i.e., force-length and force-velocity properties) can be used to predict force and work output with reasonable accuracy12,24,27. Under dynamic conditions with variable loading, the force increases as a function of velocity28 and has a complex relationship with strain and activation29,30. This contradicts the isotonic force-velocity and isometric force-length properties of muscles28. In the rat MG, strain and velocity transients are evidence of loading, such as foot contact or interaction with the environment (i.e., rough terrain, wind, sudden change in direction for predation avoidance) (Figure 9). These rat MG strain trajectories, like most realistic conditions, have sudden changes in the applied load, force production, and work output2,28. This experimental method aims to highlight these complex interactions among strain, velocity, and activation dynamics under in vivo conditions that are not well explained by traditional force-length and force-velocity relationships.
Other issues can occur when the muscle starting length is too short or long. A too-short starting length will result in a reduced rate of rise in tension during the passive and active stretch (not shown), whereas a too-long starting length will result in an increased rate of rise in passive tension (see Figure 1B). Using the ratio of active to passive tension can be helpful. For example, in rat MG, passive tension (N) is generally around half the active tension (Figure 2). If a muscle starts at too long a length and/or is stretched to a length that is too long, the passive tension may be too high relative to the active tension (see Figure 1B), and the force may decrease quickly due to overstretching. Also, stretching to a length that is too long will potentially damage the muscle and can cause the muscle to fatigue more quickly. Additionally, active tension may appear unfused if the starting length is too short and/or the muscle is not stretched to a long enough length.
Preliminary experiments are necessary to determine starting length and excursion based on L0. Additional preliminary experiments may be needed to adjust the duration of stimulation if the activation dynamics of the muscles used are different. These optimizations are needed because the fiber type composition and/or activation dynamics of in vivo and ex vivo muscles may be different. In our representative results (Figure 4 and Figure 5), we used two stimulation protocols for mouse EDL during ex vivo experiments to replicate in vivo rat MG force production. To optimize force production in the mouse EDL to best fit in vivo rat MG, stimulation duration was increased (Figure 2 and Figure 3). Rat MG is comprised of slower fiber types than mouse EDL31,32,33. This was evident in &#34;avatar&#34; experiments because ex vivo mouse EDL muscles produced force faster after excitation, and force decreased at a faster rate after deactivation than observed in vivo in rat MG15 (Figure 2), even after accounting for excitation-contraction delay differences between in vivo and ex vivo conditions34. Depending on the ex vivo and in vivo target muscles, optimization of stimulation might be needed in other &#34;avatar&#34; experiments as well. Either the mouse EDL or soleus (SOL) muscles may be used in this ex vivo work loop technique. EDL was chosen as an &#34;avatar&#34; for the rat MG due to the similarities in muscle fiber type and pennation structure. It is possible that some muscles may have a complex structure and cannot be emulated using muscles from laboratory rodents as an &#34;avatar&#34;.
While &#34;avatar&#34; experiments do need some manual optimization to best replicate in vivo force production, the technique is applicable to a variety of different animals and locomotor modes. The &#34;avatar&#34; technique can be especially useful to understand in vivo force production in animals whose muscles are too large or otherwise inaccessible for ex vivo experiments. While only preliminary work has been done on larger animals35, this work has shown potential for the applicability of this technique across animals, muscles, and locomotor gait using laboratory mice as &#34;avatars&#34;. The usefulness of &#34;avatar&#34; experiments depends on how accurately a convenient, inexpensive, readily available, and well-characterized laboratory rodent model (i.e., mouse EDL) can be used for understanding in vivo mechanics of different muscles from varying species of vertebrates. Results from preliminary &#34;avatar&#34; experiments presented here (rat MG) and elsewhere (guinea fowl LG19), suggest this technique can be used to accurately predict in vivo forces and could be applied to other animals. Future applications of this method should expand the types of muscles and animals that have been used as both targets and &#34;avatar&#34; during ex vivo and in vitro experiments. &#34;Avatar&#34; experiments allow us to examine factors that affect muscle force and work output during in vivo locomotion when muscle loading and strain vary abruptly1,2,19. Specifically, the &#34;avatar&#34; method allows us to examine the effects of strain and velocity transients on muscle force that are not captured by traditional muscle models or sinusoidal work loop experiments.

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.

<table><tbody><tr><td>Braided Non-Absorbable Silk Suture 4-0&amp;nbsp;</td><td>Mersilk&amp;nbsp;</td><td>734H</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>1086436</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Dextrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9434</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>PHR1428</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Hydorchloric Acid (HCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1.37055</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>LabView Data Collection&amp;nbsp;</td><td>Lab-View</td><td></td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M7506</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Potassium Chloride (KCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3911</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>5.43841</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>S88 Stimulator</td><td>Grass</td><td>M643H05</td><td>Available for purchase on Ebay</td></tr><tr><td>Series 300B Lever System</td><td>Aurora</td><td>1200A</td><td>includes water-jacket tissue bath</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9888</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S5881</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Wild Type Mice</td><td>Jackson Laboratory</td><td>B6C3Fe a/a Ttn mdm/J</td><td></td></tr></tbody></table>

"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.

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 (mouse EDL 12.4 + 2.12&#176;22, rat MG 20&#176; used in this study15). Scaled representative work loops of rat MG15 were compared to ex vivo &#34;avatar&#34; experiments (Figure 4) using two different stimulation protocols (one from measured EMG activity and one optimized as in step 3.3). R2 values presented here were calculated using the entire scaled stretch-shortening cycle (2 cycles/condition), with each cycle having more than 2000 points corresponding to the locomotor speed (walk = 5521 points, trot = 5002, gallop = 2502 points). Work loops were scaled to account for differences in muscle size, P0, and PCSA. Scaling was done by linearly mapping force and strain onto a similar scale (0-1) to compare rat MG and mouse EDL. Visually, it is apparent that optimizing the stimulation protocol (Figure 4B) to account for different activation dynamics of the mouse EDL and rat MG muscles improves the fit to the in vivo rat MG force compared to the EMG-based activation (see Discussion section). For the mouse EDL, approximately doubling the stimulation duration for slower strain trajectories (walk and trot) increased the R2 by 62% in walking and 109% in trot. For the faster strain trajectory (gallop), increasing stimulation time by half the observed time increased the R2 by 22%.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65610/65610fig04.jpg" /> 
Figure 4: Comparison of in vivo and ex vivo work loops. Work loops of in vivo rat MG (black) and ex vivo mouse EDL (green) during walking (2.9Hz) using in vivo strain trajectories. The thicker line indicates stimulation in both in vivo and ex vivo work loops. (A) Work loop of in vivo rat MG (black) and ex vivo mouse EDL (dashed green) during walking using EMG-based stimulation protocol. (B) Work loop of in vivo rat MG (black) and ex vivo mouse EDL (solid green) during walking (2.9Hz) using optimized stimulation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
High R2 between mouse EDL ex vivo force production and in vivo force production of rat medial gastrocnemius (MG)15 indicates good replication (Figure 5). In EMG-based stimulation experiments, average R2 values were 0.535, 0.428, and 0.77 for walk, trot, and gallop, respectively. In optimized stimulation experiments, average R2 values were 0.872, 0.895, and 0.936 in walk, trot, and gallop, respectively. As previously discussed (step 3.3, Figure 5), depending on the activation dynamics of the muscles used, the stimulation protocol may also need to be optimized. Prediction of in vivo MG force using ex vivo mouse EDL was improved across all locomotor speeds by optimizing stimulation, increasing R2 (Figure 5A,B), and decreasing root mean square error (RMSE). RMSE decreased after optimization for all speeds (Figure 6). Averaged RMSE for EMG-based stimulation was 0.31, 0.43, and 0.158 for walk, trot, and gallop. Averaged RMSE for optimized stimulation was 0.181, 0.116, 0.101 for walk, trot, and gallop.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65610/65610fig05.jpg" /> 
Figure 5: R2 Values for in vivo and ex vivo force production: Box and whisker plot of R2 values for in vivo and ex vivo force comparisons. Individual observations plotted, median, 25th, and 75th percentile indicated. (A) R2 values for in vivo and ex vivo force production using stimulation protocol based on measured in vivo EMG signal during walking at 2.9 Hz (green), trotting at 3.2 Hz (magenta), and galloping at 6.2 Hz (cyan). (B) R2 values for in vivo and ex vivo force production using optimized stimulation (see Figure 2). Optimizing the stimulation onset and duration increased R2 for all gaits. EMG-based stimulation: walk R2 = 0.50-0.55, trot R2 = 0.37-0.47, gallop R2= 0.62-0.90; optimized stimulation: walk R2 = 0.74-0.93, trot R2 = 0.85-0.92, gallop R2 = 0.87-0.97. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65610/65610fig06.jpg" /> 
Figure 6: Root-mean square error (RMSE) for in vivo and ex vivo force production. Box and whisker plot of RMSE values for in vivo and ex vivo force comparisons. Individual observations plotted, median, 25th, and 75th percentile indicated. (A) RMSE values for in vivo and ex vivo force production using EMG-based stimulation protocol. (B) RMSE values for in vivo and ex vivo using optimized stimulation protocol. Optimizing the stimulation onset and duration reduced RMSE for all gaits. Walking at 2.9 Hz (green), trot at 3.2 Hz (magenta), and gallop at 6.4 Hz (cyan). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To test the performance of traditional work loop methods at predicting in vivo muscle forces, sinusoidal work loops were also performed for the mouse EDL at the same frequency, length excursion, starting length, stimulation onset, and duration as for the &#34;avatar&#34; experiments using in vivo rat MG strain trajectories. R2 values were significantly lower than for the in vivo strain trajectories for both EMG-based and optimized stimulation protocols (Figure 7). Averaged R2 values for EMG-based stimulation using sinusoidal length trajectories were 0.062, 0.067, and 0.141 at walk, trot, and gallop frequencies. Averaged R2 values for optimized stimulation using sinusoidal length trajectories were 0.09, 0.067, and 0.141 at walk, trot, and gallop frequencies.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65610/65610fig07.jpg" /> 
Figure 7: R2 Values for in vivo and ex vivo force production using sinusoidal length changes. Box and whisker plot of RMSE values for in vivo and ex vivo force comparisons. Individual observations plotted, median, 25th, and 75th percentile indicated. R2 values for walk (green, 2.9 Hz), trot (magenta, 3.2 Hz), and gallop (cyan, 6.2 Hz) using sinusoidal length changes with EMG-based (translucent) and optimized (opaque) stimulation protocols. For both EMG-based and optimized stimulation, the R2 values were lower for the sinusoidal length changes than for in vivo length changes. EMG-based stimulation: walk R2 = 0.00 - 0.30, trot R2 = 0.00 - 0.02, gallop R2= 0.03 - 0.07; optimized stimulation: walk R2 = 0.02 - 0.21, trot R2 = 0.02 - 0.12, gallop R2 = 0.12 - 0.17. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Work loops produced by the ex vivo mouse EDL muscle using sinusoidal length trajectories do not as accurately emulate in vivo rat MG force compared to in vivo strain trajectories (Figure 8). The change in work produced by sinusoidal vs. in vivo strain trajectories can be explained by the absence of strain and velocity transients in the sinusoidal trajectory (Figure 9). While the muscles were stimulated at similar lengths during the active shortening phase of the contractions in both sinusoidal trajectories and in vivo-based strain trajectories, the onset of stimulation occurred at different phases of the cycle (e.g., stimulation onset occurred at a phase of 74% for trot EMG-based stimulation, but at a phase of 43% for walking EMG-based stimulation; see Discussion section).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65610/65610fig08.jpg" /> 
Figure 8: Comparing in vivo and ex vivo sinusoidal&#160;work loops. (A) In vivo work loop (black) from rat MG and ex vivo work loop (dashed magenta) from mouse EDL using sinusoidal strain trajectory and EMG-based stimulation. (B) In vivo work loop (black) from rat MG and ex vivo work loop (solid magenta) from mouse EDL using sinusoidal strain trajectory and optimized stimulation. Note that the sinusoidal work loops overestimate the in vivo work due to the absence of strain and velocity transients in the sinusoidal trajectory. EMG-based stimulation R2 = 0.0003, optimized stimulation R2 = 0.084. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65610/65610fig09v2.jpg" /> 
Figure 9:&#160;Comparison of in vivo strain and ex vivo sinusoidal length trajectories. Comparison of in vivo strain and ex vivo sinusoidal length trajectories at walk (green), trot (magenta), and gallop (blue). The solid line is in vivo strain trajectory. Dashed line ex vivo sinusoidal length trajectory. The highlighted portion is stimulation. Stimulation started at the same length during the shortening phase of the stride. Arrows indicating strain and velocity transients. Deviations from sinusoidal are impedance from outside forces on muscle. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/65610fig09largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Program used to collect isometric maximal force at optimal length. The program used to determine optimal length during supramaximal and submaximal twitch and tetanic stimulation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/NewFigure2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Viable twitch response. Twitch response of mouse EDL. Twitch force rises and falls quickly and should reach active tension of ~1 V. &#34;Noise&#34; should be minimal after the peak active tension has been reached. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/NewFigure3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Program used to collect work loop data. The program used to control muscle length of and timing of stimulation in ex vivo work loops. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/NewFigure4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Coding File 1: MATLab code used to segment and create an experimental protocol for the work loop. MATLab code that was used to segment target step information (length, EMG activation, and force) into individual strides. Code includes scaling and interpolating target animal steps into lengths that ex vivo mouse EDL can stretch. Additionally, includes code to smooth EMG signal and compare activation to select onset and duration of stimulation in ex vivo work loop experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65610/NewFigure1.pdf" target="_blank">Please click here to download this File.</a>

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

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

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.

"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 ...

avatar-modified-ex-vivo-work-loop-experiments-using-vivo-strain

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

714 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 

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Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

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Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

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Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

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

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A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures