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

Summary

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.

Abstract

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.

Introduction

Loss of motor function following traumatic peripheral nerve injury has a significant impact on the quality of life and socioeconomic status of patients^1,2,3. 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 superior^5,6. The associated donor site morbidity and limited availability of autologous nerve grafts have imposed the need for alternative techniques^7,8.

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 options^8,9. 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 costs^11,12. 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 results^{14,15,16,17,18}.

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 function^14,15. Regenerating nerve fibers might not make appropriate connections which can cause an overestimation of the number of functional connections^14,15,19,20. The best and clinically most relevant measurement to demonstrate correct reinnervation of end organs remains assessment of muscle function^21,22,23. 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 studies^{21,24,25,26,27,28}. The walking track analysis quantifies the sciatic functional index (SFI) based on measurements of pawprints from walking rats^21,29. 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 recovery^30,31.

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 repair^{34,35,36,37,38,39}. 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 properties^40,41,42,43. 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.

Protocol

All animal procedures were performed with approval of the Institutional Animal Care and Use Committee (IACUC A334818).

1. Calibration of the force transducer

1. 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.
2. 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).
3. 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.
4. Attach the clamp, force transducer and adjustable lever arm combination vertically to the testing platform using its vacuum base.
5. Fasten a hook or loop to the clamp for the calibration weights.
6. Turn on the computer and open the software (e.g., LabVIEW).
7. 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.
8. Run the VI by pressing the white arrow in the left upper corner and select New calibration. A new window will open.
9. Start the calibration process with zero weight (only the clamp with an attached hook or loop) and press OK.
10. Consecutively, add 10, 20, 30 and 50 grams of weight and press OK in between each weight measurement.
11. Once all five measurements are collected, click on Process.
12. Only accept the values if the graph on the VI displays a positive linear curve (Figure 3).
13. 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.
14. Click on Zero and the window will automatically close.

2. Animal subjects

1. 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 ITF^{10,32,45,46,47}.

3. Surgical preparation

1. Prepare all required surgical instruments prior to surgery (Table of Materials).
2. Weigh the animals to determine the required amount of anesthesia.
3. Induce anesthesia by placing the rat in a chamber gassed with 3% isoflurane in oxygen.
4. 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.
5. 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.
6. Carefully shave the hind limbs of the rat using electric clippers.
7. Place the rat in prone position on a heating pad to maintain the body temperature at 37 °C. Optionally, the body temperature can be monitored using a rectal thermometer.
8. 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.
9. 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.

4. Surgical approach to the common peroneal nerve

1. Place the rat in either the right or left lateral recumbent position depending on which side will be measured first.
2. 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.
3. 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.
4. 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.
5. Isolate the common peroneal nerve branch (usually the most ventral branch) of the sciatic nerve using a curved microsurgical forceps.
   ​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.

5. Dissection of the distal tibialis anterior muscle tendon

1. 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.
2. Dissect the distal TA muscle tendon from the surrounding tissue using a scalpel with a surgical blade no. 15.
3. 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.
   ​NOTE: Regularly (approximately every 5 minutes), moist the TA muscle with heated 0.9% NaCl (37 °C) to prevent cooling and desiccation.

6. Isometric tetanic force measurement

1. Connect the bipolar electrode cables and the ground cable according to their color to a bipolar stimulator device.
2. 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.
3. Transfer the animal together with the heating pad to the testing platform.
4. 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.
5. Attach a holder with a custom clamp to the testing platform using its vacuum base.
6. 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.
7. 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 °C) to prevent cooling and desiccation.
8. 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.
9. 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.
10. Optimization of the muscle length
    1. 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.
    2. Select Parameter test and turn on Trigger collection in the VI.
    3. Increase the muscle length (preload) by adjusting the lever arm attached to the force transducer.
    4. Start at 10 g of preload and use increments of 10 g until the maximum active muscle force is determined.
    5. 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.
    6. To stop the measurement, hit Trigger collection again in the VI.
    7. 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).
    8. Calculate the active muscle force by subtracting the preload from the mean peak output force.
    9. 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.
11. Measurement of isometric tetanic force
    1. After determining the ideal muscle length, let the muscle rest at zero preload for 5 minutes prior to starting the tetanic muscle contractions.
    2. Meanwhile, switch from Parameter test to Frequency test on the VI and adjust the stimulus intensity to 10 V on the bipolar stimulator device.
    3. Keep the delay and stimulus pulse duration at 2 ms and 0.4 ms, respectively.
    4. 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.
    5. Click on Trigger collection and set to the predetermined optimal muscle length.
    6. 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.
    7. 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.
    8. 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 °C) to prevent cooling and desiccation.
    9. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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