Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number RO1 NS 102360. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All animal procedures were performed with approval of the Institutional Animal Care and Use Committee (IACUC A334818).
1. Calibration of the force transducer
<ol>
	<li>Ensure that the computer is properly connected to the USB-6009 multifunctional I/O data acquisition (DAQ) device, which in turn should be connected to the force transducer. 
	NOTE: Other rat strains and species may require a different load-cell force transducer as higher forces are to be expected 44.</li>
	<li>Attach a custom clamp fashioned from a modified surgical hemostat to the force transducer that is mounted to a vacuum base adjustable lever arm. 
	NOTE: The custom-made clamp consists of a surgical hemostat modified with a tightening screw that allows for adjustment of the tension (Figure 1).</li>
	<li>Position the custom-made acrylic glass testing platform, which contains two wooden blocks for fixation of the rat hind limb, on the table. 
	NOTE: Other materials such as urethane can also be used instead of wood as long as the K-wires are able to penetrate and fixate.</li>
	<li>Attach the clamp, force transducer and adjustable lever arm combination vertically to the testing platform using its vacuum base.</li>
	<li>Fasten a hook or loop to the clamp for the calibration weights.</li>
	<li>Turn on the computer and open the software (e.g., LabVIEW).</li>
	<li>Once the software is opened, start the custom-made virtual instrument (VI) for ITF measurement (Figure 2). 
	NOTE: Figure 2 contains the LabVIEW code in a VI snippet. This VI snippet can be dragged onto the block diagram in LabVIEW. It will automatically be transformed into a graphical code. For this experiment the sampling rate was set at 2000 Hz with 25 samples to read for each iteration.</li>
	<li>Run the VI by pressing the white arrow in the left upper corner and select New calibration. A new window will open.</li>
	<li>Start the calibration process with zero weight (only the clamp with an attached hook or loop) and press OK.</li>
	<li>Consecutively, add 10, 20, 30 and 50 grams of weight and press OK in between each weight measurement.</li>
	<li>Once all five measurements are collected, click on Process.</li>
	<li>Only accept the values if the graph on the VI displays a positive linear curve (Figure 3).</li>
	<li>Reposition the clamp, force transducer and adjustable lever arm combination horizontally on the testing platform. This will be the position used for measuring the ITF.</li>
	<li>Click on Zero and the window will automatically close.</li>
</ol>
2. Animal subjects
<ol>
	<li>Use male Lewis rats weighing between 300-500 g. 
	NOTE: For comparison of nerve regeneration, it is imperative to use the same rat strain in both the control and experimental groups, since weight and incidence of autotomy are strain dependent and can tremendously influence the results of the ITF10,32,45,46,47.</li>
</ol>
3. Surgical preparation
<ol>
	<li>Prepare all required surgical instruments prior to surgery (Table of Materials).</li>
	<li>Weigh the animals to determine the required amount of anesthesia.</li>
	<li>Induce anesthesia by placing the rat in a chamber gassed with 3% isoflurane in oxygen.</li>
	<li>Deeply anesthetize the rat using a cocktail of ten-parts ketamine (100 mg/mL) and one-part xylazine (100 mg/mL) at a dosage of 1 mL/kg body weight via an intraperitoneal injection. Monitor the depth of anesthesia based on the response to a toe pinch and by observing the respiratory rate.</li>
	<li>Approximately 30 minutes after the initial dosage of the ketamine/xylazine cocktail, administer a supplementary dose of 0.3-0.6 mL/kg body weight of only ketamine (100 mg/mL) intraperitoneally to maintain adequate anesthesia throughout the entire procedure, which is defined as a low respiratory rate and an absent response to a toe pinch. 
	CAUTION: It is important to meticulously administer the required anesthesia as an overdose cannot be counteracted.</li>
	<li>Carefully shave the hind limbs of the rat using electric clippers.</li>
	<li>Place the rat in prone position on a heating pad to maintain the body temperature at 37 &#176;C. Optionally, the body temperature can be monitored using a rectal thermometer.</li>
	<li>Inject 5 mL of 0.9% sodium chloride (NaCl) subcutaneously into the loose skin over the neck of the rat to preserve an adequate hydration status throughout the procedure.</li>
	<li>Due to the non-survival nature of this procedure, the surgical field and instruments do not require to be sterile. The surgeon should use personal protective equipment (PPE) and surgical loupes are advised for proper visualization of the anatomical structures.</li>
</ol>
4. Surgical approach to the common peroneal nerve
<ol>
	<li>Place the rat in either the right or left lateral recumbent position depending on which side will be measured first.</li>
	<li>Create a 2-3 cm incision in the skin of the posterolateral thigh parallel to the femur starting at the greater trochanter using a surgical no. 15 blade.</li>
	<li>Identify the plane between the biceps femoris muscle and the gluteus maximus and vastus lateralis muscles and perform a blunt dissection using tenotomy scissors to separate these muscles and expose the underlying sciatic nerve.</li>
	<li>Locate the trifurcation of the sciatic nerve and place a retractor to acquire better access. The three branches of the sciatic nerve include the common peroneal nerve, the tibialis nerve and the sural nerve.</li>
	<li>Isolate the common peroneal nerve branch (usually the most ventral branch) of the sciatic nerve using a curved microsurgical forceps. 
	&#8203;NOTE: In case of uncertainty, gently stimulate the isolated nerve with a surgical nerve stimulator and observe the motor response. Stimulation of the common peroneal nerve results in dorsiflexion of the paw.</li>
</ol>
5. Dissection of the distal tibialis anterior muscle tendon
<ol>
	<li>In order to expose the TA muscle and its insertion, incise the skin at the anterolateral aspect of the lower leg, starting at the knee joint and descending to the mediodorsal side of the hind paw.</li>
	<li>Dissect the distal TA muscle tendon from the surrounding tissue using a scalpel with a surgical blade no. 15.</li>
	<li>Using a mosquito forceps, bluntly dissect the TA muscle tendon towards the insertion and cut the tendon as distal as possible. Leave the proximal TA muscle undisturbed, preserving the neurovascular pedicle. 
	&#8203;NOTE: Regularly (approximately every 5 minutes), moist the TA muscle with heated 0.9% NaCl (37 &#176;C) to prevent cooling and desiccation.</li>
</ol>
6. Isometric tetanic force measurement
<ol>
	<li>Connect the bipolar electrode cables and the ground cable according to their color to a bipolar stimulator device.</li>
	<li>Attach the other end of the bipolar electrode cables to a subminiature electrode. 
	NOTE: The reference electrode (red, anode) should be placed distal and the active electrode (black, cathode) proximal.</li>
	<li>Transfer the animal together with the heating pad to the testing platform.</li>
	<li>Fixate the hind limb of the rat to the wooden block using two 1 mm Kirschner wires through the ankle and the lateral condyle of the distal femur avoiding the posterior aspect of the knee. 
	CAUTION: Avoid vascular damage to the popliteal artery and vein which are located dorsally to the femur condyle.</li>
	<li>Attach a holder with a custom clamp to the testing platform using its vacuum base.</li>
	<li>Secure the distal TA muscle tendon to the clamp attached to the force transducer. 
	NOTE: The clamp and force transducer should be positioned parallel to the course of the TA muscle.</li>
	<li>Place the retractor at the posterolateral thigh of the rat in order to access the common peroneal nerve. 
	NOTE: The sciatic nerve and its branches should be kept moist with heated 0.9% NaCl (37 &#176;C) to prevent cooling and desiccation.</li>
	<li>Insert the ground cable in the surrounding muscles (e.g., the vastus lateralis muscle). 
	NOTE: The Grass SD9 stimulator requires a ground cable to reduce electrical artifacts. Newer stimulators might not require an extra ground cable.</li>
	<li>Hook the common peroneal nerve to the subminiature electrode and fix its position using the holder on the platform (Figure 4). 
	NOTE: Ensure that only the common peroneal nerve is hooked to the subminiature electrode.</li>
	<li>Optimization of the muscle length
	<ol>
		<li>Turn the bipolar stimulator device on and adjust the settings as follow: square monophasic pulse, delay 2 ms, stimulus pulse duration 0.4 ms, stimulus intensity 2 V. 
		NOTE: The delay determines the time between the sync out pulse and the delivery of the leading edge of the pulse.</li>
		<li>Select Parameter test and turn on Trigger collection in the VI.</li>
		<li>Increase the muscle length (preload) by adjusting the lever arm attached to the force transducer.</li>
		<li>Start at 10 g of preload and use increments of 10 g until the maximum active muscle force is determined.</li>
		<li>For each preload, apply two single twitches directly after each other using the button on the bipolar stimulator device. The output will be visible on the screen and the rat should show dorsiflexion of the paw. 
		NOTE: Before stimulating the nerve, always remove any excess 0.9% NaCl surrounding the nerve using cotton tipped applicators to ensure the signal is not conducted to the surrounding tissue.</li>
		<li>To stop the measurement, hit Trigger collection again in the VI.</li>
		<li>If the program automatically detects the two peak output forces click on Accept. In case the program does not automatically select these output forces, press Decline and select the peaks manually. The two peak output forces will be averaged to a mean peak output force (Figure 5).</li>
		<li>Calculate the active muscle force by subtracting the preload from the mean peak output force.</li>
		<li>Write down the active force for each preload to visualize the trend and recognize the maximum active force (Figure 6). A spreadsheet can also be used.</li>
	</ol>
	</li>
	<li>Measurement of isometric tetanic force
	<ol>
		<li>After determining the ideal muscle length, let the muscle rest at zero preload for 5 minutes prior to starting the tetanic muscle contractions.</li>
		<li>Meanwhile, switch from Parameter test to Frequency test on the VI and adjust the stimulus intensity to 10 V on the bipolar stimulator device.</li>
		<li>Keep the delay and stimulus pulse duration at 2 ms and 0.4 ms, respectively.</li>
		<li>Measure the isometric tetanic muscle force using increasing stimulus frequencies starting at 30 Hz with increments of 30 Hz until the maximum force plateau is observed.</li>
		<li>Click on Trigger collection and set to the predetermined optimal muscle length.</li>
		<li>Press the Repeat button on the bipolar stimulator device to induce a tetanic stimulation for a maximum of 5 seconds or until a force peak is clearly observed. 
		NOTE: Before stimulating the nerve, always remove any excess 0.9% NaCl surrounding the nerve using cotton tipped applicators to ensure the signal is not conducted to the surrounding tissue.</li>
		<li>To collect the data, press Trigger collection again and document the maximum output force. In case the program does not automatically detect the peak maximum output force, press Decline and select the peak manually.</li>
		<li>Let the muscle rest again at zero preload for 5 minutes prior to starting the next tetanic muscle contractions. 
		NOTE: Regularly (approximately every 5 min), moist the TA muscle with heated 0.9% NaCl (37 &#176;C) to prevent cooling and desiccation.</li>
		<li>Continue increasing the stimulus frequency until the maximum force plateau is reached. The force plateau will be defined as the maximum isometric tetanic force. 
		NOTE: After this step, remove the K-wires, staple or suture the skin and repeat the entire procedure to the contralateral hind limb, starting at step 4.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol describes a previously validated method for acquiring accurate maximum ITF measurements of the TA muscle in the rat model32. The recovery of maximum strength after experimental nerve reconstruction treatments is of primary interest in the clinical setting as it proves that the nerve not only regenerated, but also made working connections with the target muscle. The ITF can be used in a small nerve gap model, such as the rat sciatic nerve model32, and with a few modifications to the protocol, it can also be used in a larger nerve gap rabbit model33. 
There are several critical steps that should be considered to ensure consistent and reliable maximum isometric muscle force measurements. The importance of carefully selecting the type of anesthesia to prevent skeletal muscle side effects has previously been described32,33. The use of isoflurane has demonstrated a time dependent decrease in muscle force, which can be explained by its ability to induce sarcoplasmic reticulum stimulated release of calcium33,48. The effect of ketamine/xylazine on the muscle force has proven to be minimal based on our experience and previous study32. Secure attachment of the distal TA muscle tendon to the force transducer is also of great importance for accurate measurements. Slippage or tearing of the tendon should be prevented or directly corrected. Therefore, a custom-made clamp was created from a surgical hemostat and modified with a tightening screw. Other research groups have described a technique of drying the tendon for about 30 minutes to mechanically strengthen the interface between the tendon and a clamp49. In order to maintain endurance of the muscle it is critical to avoid desiccation of the TA muscle and tendon with warm 0.9% NaCl and implement a 5-minutes resting period between each tetanic stimulation. The resting period is based on the activity of the phosphagen system, also known as the immediate energy source, which is important for explosive muscle contractions. It consists of adenosine triphosphate (ATP) and creatine phosphate activity and provides energy for less than 10 seconds of maximal activity. It requires approximately 3-5 minutes to replenish 100% of the phosphagens50. 
We recognize the limitations of the method described in this video. The non-survival nature of the procedure does not allow for serial measurements over time. Additionally, it is a detailed and time-consuming testing protocol. During the 1 to 2 hour testing time, the nerve and muscle undergo a significant number of stimulations which may result in muscle fatigue with potential decrease in ITF. This has, however, proven to be less prominent in the rat model compared to the rabbit33. 
In conclusion, the ITF measurement described in this video is an invaluable tool in experimental peripheral nerve studies to quantify motor recovery. When presented with other outcome measures such as electrophysiology and histomorphometry, a global assessment of nerve function can be provided.

Loss of motor function following traumatic peripheral nerve injury has a significant impact on the quality of life and socioeconomic status of patients1,2,3. The prognosis of this patient population remains poor due to minimal improvements in surgical techniques over the years4. Direct end-to-end tension-free epineural repair forms the gold standard surgical reconstruction. However, in cases with extended nerve gaps interposition of an autologous nerve graft has proven to be superior5,6. The associated donor site morbidity and limited availability of autologous nerve grafts have imposed the need for alternative techniques7,8.
Experimental animal models have been used to elucidate the mechanism of peripheral nerve regeneration and to evaluate outcomes of a variety of reconstructive and pharmacological treatment options8,9. The rat sciatic nerve model is the most frequently used animal model10. Their small size makes them easy to handle and house. Due to their superlative neuroregenerative potential, the diminished time between intervention and evaluation of outcomes can result in relatively lower costs11,12. Other advantages of its use include morphological similarities to human nerve fibers and the high number of comparative/historic studies13. Although the latter should be approached cautiously, as a wide variety of different outcome measures between studies makes it difficult to compare results14,15,16,17,18.
Outcome measures to assess nerve regeneration range from electrophysiology to histomorphometry, but these methods imply a correlation but do not necessarily directly measure the return of motor function14,15. Regenerating nerve fibers might not make appropriate connections which can cause an overestimation of the number of functional connections14,15,19,20. The best and clinically most relevant measurement to demonstrate correct reinnervation of end organs remains assessment of muscle function21,22,23. Creating motor function assessment tools for animal models is, however, challenging. Medinaceli et al. first described the walking track analysis, which has since been the most frequently used method to evaluate functional recovery in experimental peripheral nerve studies21,24,25,26,27,28. The walking track analysis quantifies the sciatic functional index (SFI) based on measurements of pawprints from walking rats21,29. Major limitations of the walking track analysis, such as toe contractures, automutilation, smearing of the print and poor correlation with other measures of reinnervation, have necessitated the use of other parameters for quantification of functional recovery30,31.
In previous studies in Lewis rats32 and New Zealand rabbits33, we validated the isometric tetanic force (ITF) measurement for the tibialis anterior (TA) muscle and demonstrated its effectiveness in the evaluation of muscle recovery after different types of nerve repair34,35,36,37,38,39. The TA muscle is well suited because of its relatively large size, innervation by the peroneal branch of the sciatic nerve and well elucidated biochemical properties40,41,42,43. When muscle length (preload force) and electrical parameters are optimized the ITF provides a side-to-side variability of 4.4% and 7.5% in rats32 and rabbits33, respectively.
This article provides a detailed protocol of the ITF measurement in the rat sciatic nerve model, including a thorough description of the necessary pre-surgical planning, surgical approach and dissection of the common peroneal nerve and the distal TA muscle tendon. Using predetermined values for the stimulus intensity and duration, the optimal muscle length and stimulus pulse frequency will be defined. With these four parameters, the ITF can subsequently be consistently and accurately measured.

<table><tbody><tr><td>0.9% Sodium Chloride</td><td>Baxter Healthcare Corporation, Deerfield, IL, USA</td><td>G130203</td><td></td></tr><tr><td>1 mm Kirshner wires</td><td>Pfizer Howmedica, Rutherford, NJ</td><td>N/A</td><td></td></tr><tr><td>Adson Tissue Forceps</td><td>ASSI, Westbury, NY, USA</td><td>MTK-6801226</td><td></td></tr><tr><td>Bipolar electrode cables</td><td>Grass Instrument, Quincy, MA</td><td>N/A</td><td></td></tr><tr><td>Bipolar stimulator device</td><td>Grass SD9, Grass Instrument, Quincy, MA</td><td>N/A</td><td></td></tr><tr><td>Cotton-tip Applicators</td><td>Cardinal Health, Waukegan, IL, USA</td><td>C15055-006</td><td></td></tr><tr><td>Curved Mosquito forceps</td><td>ASSI, Westbury, NY, USA</td><td>MTK-1201112</td><td></td></tr><tr><td>Force Transducer MDB-2.5</td><td>Transducer Techniques, Temecula, CA</td><td>N/A</td><td></td></tr><tr><td>Gauze Sponges 4x4</td><td>Covidien, Mansfield, MA, USA</td><td>2733</td><td></td></tr><tr><td>Ground cable</td><td>Grass Instrument, Quincy, MA</td><td>N/A</td><td></td></tr><tr><td>Isoflurane chamber</td><td>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>Ketamine</td><td>Ketalar, Par Pharmaceutical, Chestnut, NJ</td><td>42023-115-10</td><td></td></tr><tr><td>LabView Software</td><td>National Instruments, Austin, TX</td><td></td><td></td></tr><tr><td>Loop</td><td>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>Microsurgical curved forceps</td><td>ASSI, Westbury, NY, USA</td><td>JFA-5B</td><td></td></tr><tr><td>Microsurgical scissors</td><td>ASSI, Westbury, NY, USA</td><td>SAS-15R-8-18</td><td></td></tr><tr><td>Microsurgical straight forceps</td><td>ASSI, Westbury, NY, USA</td><td>JF-3</td><td></td></tr><tr><td>Retractor</td><td>ASSI, Westbury, NY, USA</td><td>AG-124426</td><td></td></tr><tr><td>Scalpel Blade No. 15</td><td>Bard-Parker, Aspen Surgical, Caledonia, MI, USA</td><td>371115</td><td></td></tr><tr><td>Slim Body Skin Stapler</td><td>Covidien, Mansfield, MA, USA</td><td>8886803512</td><td></td></tr><tr><td>Subminiature electrode</td><td>Harvard Apparatus, Holliston, MA</td><td>N/A</td><td></td></tr><tr><td>Surgical Nerve Stimulator</td><td>Checkpoint Surgical LCC, Cleveland, OH, USA</td><td>9094</td><td></td></tr><tr><td>Terrell Isoflurane</td><td>Piramal Critical Care Inc., Bethlehem, PA, USA</td><td>H961J19A</td><td></td></tr><tr><td>Testing platform</td><td>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>Tetontomy Scissors</td><td>ASSI, Westbury, NY, USA</td><td>ASIM-187</td><td></td></tr><tr><td>Traceable Big-Digit Timer/Stopwatch</td><td>Fisher Scientific, Waltham, MA, USA</td><td>S407992</td><td></td></tr><tr><td>USB-6009 multifunctional I/O data acquisition (DAQ) device</td><td>National Instruments, Austin, TX</td><td>779026-01</td><td></td></tr><tr><td>Vacuum Base Holder</td><td>Noga Engineering &amp;amp; Technology Ltd., Shlomi, Isreal</td><td>N/A</td><td>Attached clamp is custom-made</td></tr><tr><td>Weight (10 g)</td><td>Denver Instruments, Denver, CO, USA</td><td>820010.4</td><td></td></tr><tr><td>Weight (20 g)</td><td>Denver Instruments, Denver, CO, USA</td><td>820020.4</td><td></td></tr><tr><td>Weight (50 g)</td><td>Denver Instruments, Denver, CO, USA</td><td>820050.4</td><td></td></tr><tr><td>Xylazine</td><td>Xylamed, Bimeda MTC Animal Health, Cambridge, Canada</td><td>1XYL002</td><td></td></tr></tbody></table>

maximum isometric tetanic force measurement of the tibialis anterior muscle in the rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Five parameters are used to measure the ITF measurement. These include muscle tension (preload force), stimulus intensity (voltage), stimulus pulse frequency, stimulus duration of 0.4 ms and a delay of 2 ms. Prior to measuring the ITF, the optimal muscle tension has to be determined using two single twitch muscle contractions at an intensity of 2 V during the parameter test. These stimuli cause dorsiflexion of the paw and produce an output signal on the graph in the VI (Figure 5). These single twitch curves ideally have a rapid vertical upswing representing the contraction period directly followed by a slower vertical decrease period demonstrating the relaxation period. The program will average these two peak output forces, but the active force has to be manually calculated by subtracting the preload force from the mean output force. In the example in Figure 5, a preload of 10 g results in two peak output forces of 411.09 g (4.03 N) and 379.78 g (3.73 N), which is averaged to a mean peak output force of 395.43 g (3.88 N). When the active forces of each preload are plot in a graph, the maximum active force can be identified. These active forces usually produce a bell-shaped curve and the maximum active force for Lewis rats weighing 300-500 g should be around 30-40 g (0.29-0.39 N) (Figure 6).
For the tetanic stimulations during the frequency test, the stimulus intensity is increased to a supra-maximal voltage (10 V) to ensure maximal activation of all TA muscle motor units using increasing frequencies. The optimal tetanic curve increases and decreases sharply and has a slowly decreasing plateau phase with minimal oscillations. Figure 7 depicts an example of a tetanic curve at a stimulus frequency of 30 Hz with an isometric tetanic force of 803.25 g (7.88 N). The highest force plateau is defined as the maximum ITF.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61926/61926fig01.jpg" /> 
Figure 1: Image of customized clamp fashioned from a surgical hemostat and modified with a tightening screw that allows for adjustment of the tension. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61926/61926fig02.jpg" /> 
Figure 2: Graphical code for virtual instrument for isometric tetanic force measurement on LabVIEW. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61926/61926fig03.jpg" /> 
Figure 3: Calibration of the force transducer. Successful calibration of the force transducer with five weights (0, 10, 20, 30 and 50 g) should result in a positive linear curve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61926/61926fig4v2.jpg" /> 
Figure 4: Schematic overview of experimental setup for isometric tetanic force measurement. (Copyrighted and used with permission of the Mayo Foundation for Medical Education and Research; all rights reserved. Reprinted from: Shin, R. H.&#160;et al. Isometric tetanic force measurement method of the tibialis anterior in the rat. Microsurgery. 28 (6), 452-457 (2008)). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61926/61926fig05.jpg" /> 
Figure 5: Representative single twitch curves for optimization of muscle length. For each preload measurement, two single twitches are applied. These single twitch curves have a rapid vertical upswing (contraction period) followed by a vertical decrease (relaxation period). The two peak output forces will be averaged to a mean peak output force. In this example with a Lewis rat, a preload of 10 g results in two peak output forces of 411.09 g (4.03 N) and 379.78 g (3.73 N), which is averaged to a mean peak output force of 395.43 g (3.88 N). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig05large.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/61926/61926fig06.jpg" /> 
Figure 6: Optimal muscle length (preload). The active muscle force can be calculated by subtracting the preload from the mean peak output force. The active muscle force for each preload should be documented until a drop in active muscle force is visible. The preload yielding the highest active muscle force will be used to measure the isometric tetanic force. The optimal preload for Lewis rats weighing 300-500 g should be around 30-40 g (0.29-0.39 N) (N=10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61926/61926fig07.jpg" /> 
Figure 7: Representative isometric tetanic force curve. The optimal tetanic curve increases sharply, then has a slowly decreasing plateau phase followed by a sharp decrease. The highest force plateau is defined as the maximum ITF. This example depicts the tetanic curve at a stimulus frequency of 30 Hz with an isometric tetanic force of 803.25 g (7.88 N). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61926/61926fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat at JoVE.com

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

maximum-isometric-tetanic-force-measurement-tibialis-anterior-muscle

Nanyang Technological University

Research

JoVE Journal

Neuroscience

4.6K Views.  Mayo Clinic. Het algemene doel van deze video is om een stapsgewijze instructie te geven over het meten van de isometrische tetanische kracht van de tibialis voorste spier in de rat, een reproduceerbare en gevalideerde methode om functioneel herstel na ischiaszenuwletsel of reconstructie te beoordelen. Het model van de rattensciatische zenuwdefect wordt vaak gebruikt om experimentele behandelingsopties voor perifere zenuwbeschadiging te evalueren. Om de gouden standaardbehandeling voor segmentale zenuwdef...

Video: Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Medicine

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Bioengineering

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Biology

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

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