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</sup...

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 fe...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 sing...

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 ...

The overall goal of this video is to provide a step-by-step instruction on how to measure the isometric tetanic force of the tibialis anterior muscle in the rat, a reproducible and validated method to assess functional recovery after sciatic nerve injury or reconstruction. The rat sciatic nerve defect model is often used to evaluate experimental treatment options for peripheral nerve injury. In order to replicate the gold standard treatment for segmental nerve defects in rats, usually a 10 millimeter segment of the sciatic nerve is excised reversed in place as an interposition autograft.In experimental treatments, the nerve gap can be reconstructed using other techniques, such as a nerve allograft or conduit. For clinical translation of these experimental techniques, it is important to measure the extent of motor function recovery in the target muscle. In this video, we will describe how to do that for the tibialis anterior muscle by stimulating the common peroneal nerve.Start the procedure by calibrating the force transducer. Position the testing platform, which contains two wooden blocks on the procedure table. Make sure the computer is connected to a data acquisition device, which in turn should be connected to the force transducer.Attach the force transducer vertically to the testing platform using its vacuum base. Fasten the loop to the clamp for the calibration weights. Open the software and run the virtual instrument for isometric tetanic force measurement and select new calibration.The calibration starts with zero weight on the loop. Consecutively, with 10, 20, 30, and 50 grams of weight and collect the data in between each weight measurement. Select process once all five weight measurements are collected.In case of a successful calibration, the graph should depict a positive linear curve, accept the values to continue. Remove the loop and the weights from the clamp and reposition the force transducer horizontally on the testing platform. Select zero to finish the calibration process.After inducing anesthesia using an isoflurane chamber, shave a hindlimbs and place the rat on a 37 degree heating pad. Deeply anesthetized the rat with an intraperitoneal injection of a ketamine and xylazine cocktail and administer five to six milliliters of saline subcutaneously to maintain an adequate hydration status throughout the entire procedure. Place the rat on its side and create a 2 to 3 centimeter posterolateral thigh incision, parallel to the femur.In order to expose the peroneal nerve branch of the sciatic nerve, separate the biceps femoris muscle from the gluteus maximus and vastus lateralis muscle by dissecting the fascial plane between these muscles. Place the retractor between the separated muscles to have better access to the sciatic nerve. Identify the trifurcation of the sciatic nerve into the peroneal nerve, the tibialis nerve, and the sural nerve.Use a curved forceps to dissect the peroneal nerve branch from the surrounding tissue. In case of uncertainty, gently pinch the nerve and observe whether the motor response is dorsiflexion of the hind paw. To expose the tibialis anterior muscle, place the rat on his back and incise the anterolateral aspect of the lower limb, starting at the knee joint and descending to the mediodorsal side of the paw.Isolate the distal tibialis anterior muscle tendon from the surrounding tissue. Use a mosquito forceps to bluntly dissect the tendon all the way to its insertion and cut the tendon at the insertion point. The first step in the process of isometric tetanic force measurement is optimization of the muscle length, also known as the parameter test.Transfer the rat together with the heating pad to the testing platform use two 1 millimeter k-wires to fix the hindlimb to the wooden block. Drill the first k-wire through the distal femur and the second one through the ankle. Attach the holder for the subminiature electrode to the testing platform and position the clamp around the location of the peroneal nerve.Insert the ground cable into the surrounding muscles and place back the retractor for access to the peroneal nerve. Attach the bipolar electrode cables to the subminiature electrode and hook the electrode around the peroneal nerve using the curved forceps. Use the clamp on the holder to fix the position of the subminiature electrode.Clamp the distal tibialis anterior muscle tendon to the clamp which is attached to the force transducer. For accurate measurement, it is important that the force transducer is positioned parallel to the course of the tibialis anterior muscle. Make sure the rat reference cable is placed distal on the subminiature electrode and the black active electrode cable proximal.Connect both these cables and the ground cable to a bipolar stimulator device. For optimization of muscle length, adjust the settings of the bipolar stimulator to a delay of two milliseconds duration of 0.4 milliseconds and a stimulus intensity of 2 volts. Once everything is well connected, set the software on parameter test and press trigger collection.Increase the muscle length, also known as a preload, by moving the lever arm attached to the force transducer. The preload is the big kit on top of the graph on the screen. Start at a 10 gram preload and give two single twitches by pushing the stimulus button on the bipolar simulator downwards.The output will be visible on the graph as two peak output forces and the rat should show dorsiflexion of the paw. Press trigger collection again to measure these two peaks. Usually, the program automatically detects the peaks.In that case, press accept to continue. If the software, however fails to recognize the peaks, click the decline and manually select the peaks. The two output forces will be averaged to a mean peak output force, which will be presented on the right side of the graph.The active muscle force can then be calculated by subtracting the preload from this mean peak output force. Use increments of 10 grams preload until the preload that results in the maximum active muscle force is identified. Each preload measurement should be conducted in the same way by pressing the stimulus button on the bipolar simulator twice downwards.To make this process easier, write down the active muscle force for each preload and stop increasing the preload as soon as the active muscle force drops. In this example, the maximum active muscle force is switch at a preload of 20 grams. The preload yielding the highest active muscle force, in other words, the optimal muscle length, will be used to measure the isometric tetanic force.Release the tension on the muscle and moisturize the muscle with heated saline. Wait five minutes before starting the measurement of isometric tetanic force. While waiting, adjust the stimulus intensity to 10 volts, lead the duration on 0.4 milliseconds and the delay on two milliseconds.The isometric tetanic force will be measured using increasing frequencies, starting at 30 Hertz. Switch from parameter test to frequency test on the software and move the force transducer to the predetermined optimal muscle length. Now induce the tetanic stimulation by pressing the stimulus button on the bipolar simulator upwards for a maximum of five seconds or until a force peak is clearly observed.The optimal tetanic curve increases sharply, then has a slowly decreasing plateau phase followed by a sharp decrease. Document the force peak for each stimulus frequency. Let the muscle rest at zero preload for five minutes and moisturize the muscle with heated saline between each measurement.Use increments of 30 Hertz and a resolution of 10 Hertz until the maximum force plateau is observed. This will be the isometric tetanic force. Use staples or sutures to close the skin and repeat the entire procedure to the contralateral hindlimb.Euthanize the animal after finishing the measurement on the contralateral side. After watching this video, you should have a good understanding of how to reproducibly measure the isometric tetanic muscle force in rats that have undergone sciatic nerve reconstruction.

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

Abstract