The authors have no acknowledgements.

The experimental procedures outlined received Institutional ethical approval and are in accordance with the Declaration of Helsinki. Data were collected from a representative participant who was aware of the procedures and gave his written informed consent.
1. Instrument Preparation
<ol>
	<li>Clean the skin at the electrode location by shaving, and remove the dirt with alcohol to obtain low impedance (&#60;5 k&#937;).</li>
	<li>Place two AgCl surface electrodes (recording diameter of 10 mm) at 2/3 of the line between the medial condylis of the femur to the medial malleolus for the soleus muscle; on the most prominent bulge of the muscle for the medial gastrocnemius; at 1/3 of the distance along a line between the head of the fibula and the heel for the lateral gastrocnemius; and at 1/3 of the distance along a line between the tip of the fibula and the tip of the medial malleolus for the tibialis anterior muscle, with an interelectrode distance (center-to-center) of 2 cm, according to the SENIAM recommendations30. 
	Note: The soleus muscle electrodes have to be positioned under the distal insertion of gastrocnemii muscles to ensure that they are not recording activity from the heads of the gastrocnemii muscles (cross-talk).</li>
	<li>Place a reference electrode in a central position on the same leg (between stimulation and recording sites).</li>
	<li>Adjust the height and the depth of the chair to obtain an ankle angle of 90&#176; (0&#176; = full plantar flexion), so that the soleus and gastrocnemii muscles are not stretched and the H-reflex is not altered11,12.
	<ol>
		<li>Set the knee angle at 90&#176; (0&#176; = full knee extension) due to the biarticular nature of the gastrocnemii muscles. However, the optimal ankle angle to perform a maximal voluntary torque of the plantar flexors is 70-80&#176; (0&#176; = full plantar flexion)26. Thus, ankle angle will depend on the parameter of interest (electrophysiological versus mechanical recordings). 
		Note: Regardless of the chosen initial angle, it must remain constant throughout the experiment to standardize neuromuscular excitability11,12,27,28.</li>
		<li>Pay particular attention when monitoring the subjects&#8217; posture during the test to maintain constant cortico-vestibular influences on the excitability of the motor pool29.</li>
	</ol>
	</li>
	<li>Firmly strap the ankle to an ergometer, with the anatomical axis of the joint (external malleolus) aligned with the axis of rotation of the ergometer25.
	<ol>
		<li>Have the subject exert pressure on a footplate attached to the ergometer to record plantar flexor torque. Keep the foot immobile throughout the experiment so that small changes in torque can be detected.</li>
	</ol>
	</li>
	<li>Note: Under certain circumstances, the heel may lift slightly off the force plate if the foot and ankle are not secured, which may lead to an incomplete transmission of the torque against the plate. Figure 3 presents a description of the experimental setup.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52974/52974fig3.jpg" /> 
Figure 3: Experimental setup. Classical experimental setup to record electromyographic (EMG) and torque signals.
<ol start="6">
	<li>Connect the electrodes to the amplifier with cables.</li>
	<li>Set the sampling rate for torque and EMG measurements to 2-5 kHz. Record the EMG signal using an Analog-to-Digital (AD) conversion system. The signal is displayed on a monitor with a data acquisition system, which instantaneously gives values of several parameters (e.g. maximal value, peak-to-peak amplitude, duration). The spectrum of the EMG signal can range between 5 Hz and 2 kHz frequencies, but is mainly contained between 10 Hz and 1 kHz31. Thus, sampling frequency must be high enough to preserve signal shape during EMG recording. Amplify and filter EMG signals (gain = 500-100) using a bandwidth frequency between 10 Hz and 1 kHz8,21,32.</li>
	<li>Place the anode for the electrical stimulation over the patellar tendon.</li>
	<li>Determine the best stimulation site of the posterior tibial nerve to obtain an optimal soleus H reflex for a given intensity, using a hand-held cathode ball electrode in the popliteal fossa. Test several stimulation sites with the cathode ball electrode until a maximal value of the H reflex is reached.
	<ol>
		<li>Record tibialis anterior EMG activity to ensure that the common peroneal nerve is not activated to avoid influence from antagonist Ia afferents12. Set the pulse width at 1 msec to provide an optimal activation of the nervous fibers, especially afferent fibers10.</li>
	</ol>
	</li>
	<li>Place a self-adhesive AgCl cathode at the location of the stimulation site to ensure constant stimulus condition (e.g. pressure, orientation). 
	Note: All of these parameters (subject position, electrode location and stimulation site) do not change for the assessment of the different electrophysiological measurements. Only the intensity of the stimulation and the condition (rest versus contraction) vary.</li>
</ol>
2. Testing Procedures at Rest
<ol start="1">
	<li>Instruct the subject to remain relaxed and to keep his/her muscles at rest.</li>
	<li>Adjust the stimulation intensity to obtain maximal soleus H-reflex amplitude (Hmax; usual range: 20-50 mA). An M-wave of the soleus muscle can be observed at Hmax intensity. 
	Note: For repeated measurements (e.g. before and after a fatiguing protocol), the optimal intensity to obtain a Hmax response may vary during the session. As keeping a constant intensity can lead to an underestimation of Hmax amplitude, it is recommended that the experimenter regularly reevaluates Hmax intensity33.</li>
	<li>Record a minimum of 3 soleus H-reflex responses at this intensity with a minimum interval of 3 sec to avoid post-activation depression34. 
	Note: Although recording several responses is more suitable due to the particular sensitivity of the H-reflex, a single stimulation may be enough under some circumstances, for example when trying to avoid the effects of rapid recovery (e.g. during a fatiguing protocol).</li>
	<li>Increase the stimulation intensity to obtain maximal soleus M-wave amplitude (Mmax; usual range: 40-100 mA). Usually, set the increment in stimulation intensity at 2-4 mA, with an interval of 8-10 sec between two stimuli12,35. The desired intensity is reached when Mmax is obtained, and no H-reflex response can be observed.</li>
	<li>Set the final intensity to 120-150% of Mmax stimulus intensity to ensure that the M-wave attains a plateau of its maximal value. This intensity is called supramaximal intensity in the instructions below.</li>
	<li>Keep constant stimulation intensity for soleus M-wave recordings throughout the session.</li>
	<li>Record 3 soleus M-waves and 3 associated twitch torques at this intensity.</li>
</ol>
3. Testing Procedures During Voluntary Contraction
<ol start="1">
	<li>As a warm-up, ask the subject to perform 10 brief and non-fatiguing submaximal contractions of the plantar flexor muscles, with a few seconds rest between each of the contractions. At the end of the warm-up, take a minimum 1 min rest to avoid any fatiguing effects11.</li>
	<li>Continuously record triceps surae EMG activity. Recording soleus and gastrocnemii muscles allows the analysis of the behavior of different muscle typologies for a single stimulation site24.</li>
	<li>Instruct the subject to perform an isometric maximal voluntary contraction (MVC) of the plantar flexors. The subject has to push as hard as possible against the ergometer by contracting his plantar flexor muscles. Give visual feedback to the subject during the effort, and standardized verbal encouragement19. The MVC is reached when a plateau is observed.</li>
	<li>Deliver a paired stimulation (100 Hz frequency) at supramaximal intensity during the plateau of the MVC (superimposed doublet), and another paired stimulation when the muscle is fully relaxed immediately after the contraction (potentiated doublet) to evaluate the voluntary activation level. Deliver this paired stimulation through a specific device (e.g. Digitimer D185 MultiPulse Stimulator) or through a stimulation program associated with a single pulse stimulator.</li>
	<li>Instruct the subject to perform a second MVC of the plantar flexor with at least 1 min rest between each trial11. If the peak torque from the second trial is not within 5% of the first, additional trials must be performed36. The greatest torque achieved by the subject is taken as the MVC torque.</li>
</ol>
4. Data Analysis
<ol start="1">
	<li>Data Analysis at Rest
	<ol>
		<li>Select a time window including the EMG response associated with the twitch at rest (H-wave or M-wave).</li>
		<li>Measure the peak-to-peak amplitude, peak-to-peak duration, and/or the area of the waves (Figure 4A). If the amplitude is not directly provided by the software, subtract the minimum to the maximum values.
		<ol>
			<li>For the duration, measure the time frame starting from the maximal peak and ending to the minimal peak. For the area, calculate the integral of EMG signal starting from the beginning of the wave and ending to the end of the wave. 
			Note: Peak-to-peak amplitude can reflect: 1) neuromuscular transmission, 2) motor unit action potential amplitude and/or 3) temporal dispersion of motor unit action potential37. M-wave duration reflects neuromuscular propagation37.</li>
		</ol>
		</li>
		<li>For multiple trials, calculate the average of the waves. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from several trials (at least 3).</li>
		<li>Select the resting twitch.</li>
		<li>Measure the peak torque associated with the resting twitch (Figure 4B).</li>
		<li>For multiple trials, calculate the average peak torque of the resting twitches. If the average cannot be directly provided by the software, use spreadsheet software (e.g. the formula function in a spreadsheet program) to calculate this value from the several trials (at least 3).</li>
		<li>Repeat these procedures described in point 4.1.2 for the other desired parameters (contraction time or half-relaxation time). The analysis of twitch parameters provides indications as to the excitation-contraction coupling efficiency17. In particular, contraction time provides an index of contraction kinetics8, which can depend upon the chosen muscle group38.</li>
		<li>Calculate the ratio between the peak torque and the sum of M-waves using spreadsheet software (e.g. Excel), to quantify the electromechanical efficiency (Pt/M). As the mechanical responses evoked by posterior tibial nerve stimulation correspond to the activation of the triceps surae as a whole, amplitudes of soleus and gastrocnemii M-waves must be summed39.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52974/52974fig4.jpg" /> 
Figure 4: Explanation of electrophysiological and mechanical responses. (A) Measurement of peak-to-peak amplitude (mV), latency (ms) and area (mV.ms) of a typical M-wave. (B) Measurement of peak twitch torque (Nm), contraction time (ms) and half-relaxation time (msec) of a twitch. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52974/52974fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Data analysis in contraction
	<ol>
		<li>Select a 500 msec time window of soleus EMG activity during the plateau of MVC torque including the peak torque but excluding the time between the stimulation artefact and the end of the silent period of EMG. The silent period corresponds to the suppression of the ongoing voluntary EMG activity following stimulation.</li>
		<li>If the root mean square (RMS) is not directly provided by the software, calculate the RMS to quantify EMG activity using the following formula40: RMSEMG 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq1.jpg" /></li>
		<li>Measure or calculate the RMS of Mmax at rest over the duration of the wave.</li>
		<li>Calculate the RMSEMG/RMSMmax ratio using spreadsheet software. RMSEMG value and RMSMmax value have to be selected from the same muscle.</li>
		<li>Measure the maximal peak torque of the MVC from the baseline of torque at rest to the maximal value of MVC excluding the superimposed torque induced by the doublet stimulation (Figure 5).</li>
		<li>Measure the superimposed torque induced by the doublet stimulation during the MVC, from the voluntary torque value at the onset of the stimulation to the peak of the evoked response (Figure 5).</li>
		<li>Select the potentiated doublet.</li>
		<li>Measure the peak torque associated with the potentiated doublet.</li>
		<li>Calculate the voluntary activation level (VAL) using the following formula40: 
		<img alt="Equation 1" src="/files/ftp_upload/52974/52974eq2.jpg" /></li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52974/52974fig5.jpg" /> 
Figure 5: Measurement of superimposed and potentiated doublet on mechanical signal. To record the superimposed peak torque (Pts), stimulation doublet is evoked during the plateau of isometric maximal voluntary contraction (MVC). To record potentiated peak torque (PtP), stimulation doublet is evoked at rest after the offset of MVC.

The authors have nothing to disclose.

Percutaneous nerve stimulation enables the quantification of numerous characteristics of the neuromuscular system not only to understand the fundamental control of neuromotor function in healthy humans, but also to be able to analyze acute or chronic adaptations through fatigue or training17. This is very beneficial especially for fatiguing protocols, where measurements must be performed as soon as possible after exercise end to avoid the effects of rapid recovery42.
Although numerous studies have focused on the triceps surae muscles24, percutaneous nerve stimulation can be applied in other lower limb (e.g. tibialis anterior43,44, quadriceps muscles45,46) and upper limb muscles (e.g. biceps brachii32, flexor carpi radialis47, finger muscles48). However, nerve stimulation presents potential methodological limitations for some muscles. For instance, obtaining a H-reflex from biceps brachii muscle can be difficult to obtain at rest49. Furthermore, stimulating the musculocutaneous nerve over the brachial plexus leads to contraction of both agonist and antagonist muscles32, inducing the erroneous evaluation of the voluntary activation level. Recording nearby muscle activity allows the experimenter to ensure that only the target muscle is activated, or at least to limit activation of these nearby muscles. To overcome these limitations, some authors have suggested that stimulation over the muscle belly with larger electrodes can be a reliable method to evoke M-wave and twitches32,50. However, the spatial organization of axonal terminal branches within the muscle can differ between muscles. Thus, motor units activation would vary between nerve and muscle stimulation51. Nerve stimulation activates motor units according to the size principle, whereas the recruitment order during direct muscle stimulation is more dependent upon the spatial organization of muscle fibers under the stimulating electrodes50.
Monosynaptic aspects of the H-reflex allow the reliable assessment of spinal excitability with nerve stimulation. However, it must be noted that Ia-alpha motoneurons synapse can be subject to numerous cortical influences, such as subject&#8217;s attention52, visual environment53, head movements54 or even jaw clenching55. Peripheral factors can also influence response amplitude, such as afferent feedback from muscle stretch56. The posture of the subject has also to be carefully controlled for during the experiments and through experimental sessions to minimize cortico-spinal influences29. Furthermore, familiarization sessions can reduce intersession variability, especially for novice subjects57.
Besides these physiological concerns, stimulation characteristics (e.g. intensity, location) can widely influence the results. Although Mmax responses reach a plateau near maximal intensity, Hmax is obtained for a specific intensity. Thus, intensity of stimulation to obtain Hmax is more susceptible to variability with conditions. To ensure good reliability under different conditions (e.g. fresh or fatigued muscle), stimulus intensity should be set to Hmax intensity or below, when the reflex response lies in the ascending part of the recruitment curve58. Indeed, H-reflex amplitude can be altered for intensities above Hmax intensity due to the collision between reflex and antidromic volleys (Figure 2, number 3&#8217; and number 2). It is also recommended that the H-reflex amplitude be normalized to the Mmax response (H/Mmax ratio). It has been shown that this method allows for reliable inter- and intra-individual comparisons59.
In terms of inferring the nature of the motor command, although the VAL technique has been shown to be a reliable technique to assess descending commands40 and central fatigue19,60, this method presents some limitations. Indeed, some authors suggested that VAL overestimates maximal muscle activation61&#8211;63. It may not be sensitive enough to detect variations in activation levels during contractions above 90 % MVC62. Moreover, the use of paired stimulation to evaluate VAL can increase discomfort for subjects64. Despite the evaluation of maximal voluntary activation, this method does not provide information about cortico-spinal excitability. Transcranial magnetic stimulation could be used to assess changes at this level65&#8211;67.
The use of the RMSEMG/RMSMmax ratio to evaluate voluntary activation is less sensitive than the twitch interpolation technique due to greater response variability. Indeed, RMSEMG/Mmax ratio can remain constant whereas the twitch interpolation technique highlights a significant decrease in muscle activation68. However, the RMSEMG/RMSMmax ratio allows the experimenter to evaluate the activation of the different individual muscles of the same muscle group (e.g. soleus, medial gastrocnemius and lateral gastrocnemius for the triceps surae)17.
Particular attention should be taken with percutaneous nerve stimulation regarding stimulation protocol and data analysis to avoid misinterpretation and to permit a comparison between different studies. Numerous authors have previously established methodological recommendations to record and analyze data from percutaneous electrical stimulation20,29,34,59. In particular, plantar flexor muscles appear to be a difficult muscle group to contract maximally69&#8211;71. Practice is required to ensure that the participants, especially in populations with impaired neuromuscular function, are capable of high levels of voluntary activation prior to experimental testing72,73. Thus, MVC-dependent measures such as voluntary activation will represent erroneous values that likely reflect a lack of practice or an insufficient number of isometric MVC attempts rather than an impairment or limitation of neuromuscular function. A familiarization session should be performed prior to all studies using percutaneous nerve stimulation and/or maximal efforts.
Percutaneous electrical nerve stimulation can be used to evaluate neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercises. For instance, Lepers et al.74 observed a decrease in central activation (voluntary activation level) and muscular parameters (peak twitch, M-wave) of the quadriceps muscle following a prolonged cycling exercise. Following chronic exercise, Duchateau and Hainaut75 observed different effects of isometric and dynamic trainings on peak twitch torque properties, suggesting that skeletal muscle adapts differently to the kind of training programs. Electrical nerve stimulation is also useful to evaluate online adaptations of the neuromuscular system during various conditions, such as posture27 or a concurrent mental task21. This method can be used not only in fundamental research but also in the clinical domain76. Indeed, electrical nerve stimulation has been used to investigate central drive in the elderly77 and different diseases such as stroke78 or Parkinson&#8217;s disease79. Neuromuscular plasticity can also be assessed in pathological populations during therapy/retraining program80.

Percutaneous electrical nerve stimulation is widely used to assess neuromuscular function1. The basic principle consists of inducing an electrical stimulus to a peripheral motor nerve to evoke a muscular contraction. Mechanical (torque measurement) and electrophysiological (electromyographic activity) responses are simultaneously recorded. Torque, recorded at the considered joint, is assessed using an ergometer. The electromyographic (EMG) signal recorded using surface electrodes has been demonstrated to represent the activity of the muscle2. This non-invasive method is not painful and more easily implemented than intramuscular recordings. Both monopolar and bipolar electrodes can be used. The monopolar electrode configuration has been shown to be more sensitive to changes in muscle activity3, which can be useful for small muscles. However, bipolar electrodes have been shown to be more effective in improving the signal-to-noise ratio4 and are most commonly used as a method of recording and quantifying motor unit activity. The methodology described below will focus on bipolar recordings. EMG activity is an indicator of the efficacy and integrity of the neuromuscular system. The use of percutaneous nerve stimulation offers further insights into neuromuscular function, i.e. changes at muscular, spinal, or supra-spinal level (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/52974/52974fig1.jpg" /> 
Figure 1: Overview of the neuromuscular measurements. STIM: nerve stimulation. EMG: Electromyography. VAL: Voluntary Activation Level. RMS: Root Mean Square. Mmax: Maximal M-wave amplitude.
At rest, the compound muscle action potential, also called M-wave, is the short-latency response observed after stimulus artefact, and represents excitable muscle mass by the direct activation of motor axons leading to the muscle (Figure 2, number 3). M-wave amplitude increases with intensity until reaching a plateau of its maximal value. This response, called Mmax, represents the synchronous summation of all motor units and/or muscle fiber action potentials recorded under the surface EMG electrodes5. The evolution of the peak-to-peak amplitude or wave area is used to identify alterations of neuromuscular transmission6. Changes in the mechanical responses associated with the M-wave, i.e. peak twitch torque/force, may be due to alterations in muscle excitability and/or within the muscle fibres7. The association of Mmax amplitude and peak twitch torque amplitude (Pt/M ratio) provides an index of electromechanical efficiency of the muscle8, i.e. mechanical response for a given electrical motor command.
<img alt="Figure 2" src="/files/ftp_upload/52974/52974fig2.jpg" /> 
Figure 2: Motor and reflexive pathways activated by nerve stimulation. Electrical stimulation of a mixed (motor/sensory) nerve (STIM) induces a depolarization of both motor axon and Ia afferent firing. Depolarization of Ia afferents towards the spinal cord activates an alpha motoneuron, which in turn evokes an H-reflex response (pathway 1+2+3). Depending upon the stimulus intensity, motor axon depolarization evokes a direct muscular response: M-wave (pathway 3). At maximal M-wave intensity, an antidromic current is also generated (3&#39;) and collides with reflex volley (2). This collision partially or totally cancels the H-reflex response.
The H-reflex is an electrophysiological response used to assess changes in the Ia-&#945; motoneuron synapse9. This parameter can be assessed at rest or during voluntary contractions. H-reflex represents a variant of the stretch reflex (Figure 2, number 1-3). The H-reflex activates motor units monosynaptically recruited by Ia afferent pathways10,11, and can be subjected to peripheral and central influences12. The method of evoking a H-reflex is known to have a high intra-subject reliability to assess spinal excitability at rest13,14 and during isometric contractions15.
During a voluntary contraction, the magnitude of the voluntary neural drive can be assessed using the amplitude of the EMG signal, generally quantified using the Root Mean Square (RMS). RMSEMG is commonly used a means of quantifying the level of excitation of the motor system during voluntary contraction (Figure 1). Because of the intra- and inter-subject variability16, RMSEMG has to be normalized using the EMG recorded during a muscle-specific maximal voluntary contraction (RMSEMGmax). In addition, because changes in EMG signal may be due to alterations at peripheral level, normalization using a peripheral parameter such as M-wave is required to assess only the central component of EMG signal. This can be done by dividing the RMSEMG by the maximal amplitude or the RMSMmax of the M-wave. Normalization using RMSMmax (i.e. RMSEMG/RMSMmax) is the preferred method as it takes into consideration the possible change of the M-wave duration17.
Motor commands can also be evaluated by calculating the voluntary activation level (VAL). This method uses the twitch interpolation technique18 by superimposing an electrical stimulation at Mmax intensity during a maximal voluntary contraction. The extra-torque induced by stimulating the nerve is compared to a control twitch produced by identical nerve stimulation in a relaxed potentiated muscle19. To evaluate maximal VAL, the original twitch interpolation technique described by Merton18 involves a single stimulus interpolated over a voluntary contraction. Recently, the use of paired stimulation has become more popular because the evoked torque increments are larger, more readily detected, and less variable compared to single stimulation responses20. The VAL provides an index of the capacity of the central nervous system to maximally activate the working muscles21. Currently, VAL evaluated using the twitch interpolation technique is the most valuable method of assessing the level of muscle activation22. Furthermore, peak torque assessed using an ergometer is the most properly studied strength testing parameter applicable of use in research and clinical settings23.
Electrical nerve stimulation can be used in a variety of muscle groups (e.g. elbow flexors, wrist flexors, knee extensors, plantar flexors). However, nerve accessibility makes the technique difficult in some muscles groups. The plantar flexor muscles, especially triceps surae (soleus and gastrocnemii) muscles, are frequently investigated in the literature24. Indeed, these muscles are involved in locomotion, justifying their particular interest. The distance between stimulation site and recording electrodes allows the identification of the different evoked waves of the triceps surae muscles. The superficial part of the posterior tibial nerve in the popliteal fossa and the large number of spindles make it easier to record reflex responses compared to other muscles24. For these reasons, the currently presented reflex methodology focuses on the triceps surae group of muscles (soleus and gastrocnemius). The aim of this protocol is therefore to describe percutaneous nerve stimulation technique to investigate neuromuscular function in the triceps surae.

<table><tbody><tr><td>Biodex dynamometer</td><td>Biodex Medical System Inc., New York, USA</td><td></td><td>&lt;a href=&quot;http://www.biodex.com/&quot;&gt;www.biodex.com&lt;/a&gt;</td></tr><tr><td>MP150 Data Acquisition System</td><td>Biopac Systems Inc., Goleta, USA</td><td></td><td></td></tr><tr><td>Acknowledge 4.1.0 software</td><td>Biopac Systems Inc., Goleta, USA</td><td></td><td>&lt;a href=&quot;http://www.biopac.com/&quot;&gt;www.biopac.com&lt;/a&gt;</td></tr><tr><td>DS7A constant current high voltage stimulator</td><td>Digitimer, Hertfordshire, UK</td><td></td><td>&lt;a href=&quot;http://www.digitimer.com/&quot;&gt;www.digitimer.com&lt;/a&gt;</td></tr><tr><td>Silver chloride surface electrodes</td><td>Control Graphique Medical, Brie-Comte-Robert, France</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr><tr><td>1 Cable for connecting the Biodex to the MP150</td><td></td><td></td><td></td></tr><tr><td>1 Cable for connecting the Digitimer to the MP150</td><td></td><td></td><td></td></tr><tr><td>1 Cable for connecting the MP150 to the computer</td><td></td><td></td><td></td></tr></tbody></table>

assessment of neuromuscular function using percutaneous electrical nerve stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

Increasing stimulus intensity leads to a different evolution of response amplitudes between H- and M-waves. At rest, the H-reflex reaches a maximum value before being totally absent from EMG signal, while M wave progressively increases until reaching a plateau at maximal intensity (see Figure 4 for a graphical depiction of the M-wave and Figure 6 for the evolution of M-waves and H-reflex with intensity). For the soleus muscle, the latency between the stimulus onset and M-wave is about 10 msec (Figure 4A) and generally between 25 and 40 msec for H-wave. However, the latency will vary between the muscle groups and the subject&#8217;s limb length or overall height, due to the distance between the stimulation site and the muscle. When stimulating at M-max intensity, a maximal peak twitch torque will also be observed (Figure 4B). M-waves, H-reflexes and peak twitch torques will vary depending on the condition. For example, these parameters tend to increase during voluntary contraction, and decrease in the presence of fatigue17.
<img alt="Figure 6" src="/files/ftp_upload/52974/52974fig6.jpg" /> 
Figure 6: Typical recruitment curves at rest. Amplitudes of reflex responses (H-reflex, white round) and direct muscle responses (M-wave, black round) with increasing stimulus intensity. Bottom panels present typical traces at four progressively increased intensities (from A to B). (A) weak intensity, evoking only an H-reflex response. (B) Intensity providing the maximal H-wave amplitude (Hmax). (C) At intensity beyond Hmax, the collision between antidromic and reflex volleys induces a decrease in H response amplitude. (D) At Mmax intensity, H-reflex is totally cancelled and M-wave reaches a plateau. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52974/52974fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Maximal VAL is evaluated during a MVC. Figure 5 shows a superimposed torque induced by electrical stimulation during the MVC. The effect induced by stimulation reflects an incomplete recruitment of motor units and/or a submaximal discharge frequency of the motor units, and thus a deficit in voluntary activation (see the effect of stimulation in the middle of Figure 5). As previous parameters, maximal VAL varies depending on the condition (e.g. level of contraction, fatigue)21.
These different techniques have previously been validated. Indeed, recent studies demonstrated a good reliability for M wave and the associated peak twitch torque22, H-reflex14 and maximal VAL41.

Watch this Scientific Journal Video about Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation at JoVE.com

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

assessment-neuromuscular-function-using-percutaneous-electrical-nerve

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.8K Views.  Univ. Bourgogne Franche–Comté. We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). 

Video: Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Non-Invasive Electrical Brain Stimulation Montages for Modulation of Human Motor Function

Non-invasive electrical brain stimulation (NEBS) is used to modulate brain function and behavior, both for research and clinical purposes. In particular, NEBS can be applied transcranially either as direct current stimulation (tDCS) or alternating current stimulation (tACS). These stimulation types exert time-, dose- and in the case of tDCS polarity-specific effects on motor function and skill learning in healthy subjects. Lately, tDCS has been used to augment the therapy of motor disabilities in patients with stroke or movement disorders. This article provides a step-by-step protocol for targeting the primary motor cortex with tDCS and transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range. The setup of two different stimulation montages is explained. In both montages the emitting electrode (the anode for tDCS) is placed on the primary motor cortex of interest. For unilateral motor cortex stimulation the receiving electrode is placed on the contralateral forehead while for bilateral motor cortex stimulation the receiving electrode is placed on the opposite primary motor cortex. The advantages and disadvantages of each montage for the modulation of cortical excitability and motor function including learning are discussed, as well as safety, tolerability and blinding aspects.

Non-invasive electrical brain stimulation can modulate cortical function and behavior, both for research and clinical purposes. This protocol describes different brain stimulation approaches for modulation of the human motor system.

Non-invasive electrical brain stimulation (NEBS) is used to modulate brain function and behavior, both for research and clinical purposes. In particular, ...

Behavior

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Medicine

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

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 ...

Bioengineering

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

SFEMG in Rodents: Assessing NMJ Function in Health and Disease

Stimulated Single Fiber Electromyography (SFEMG) for Assessing Neuromuscular Junction Transmission in Rodent Models

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. Single fiber electromyography (SFEMG) is a clinically relevant and sensitive technique that measures single muscle fiber action potential responses during voluntary contractions or nerve stimulations to assess NMJ transmission. The assessment and quantification of NMJ transmission involves two parameters: jitter and blocking. Jitter refers to the variability in timing (latency) between consecutive single-fiber action potentials (SFAPs). Blocking signifies the failure of NMJ transmission to initiate an SFAP response. Although SFEMG is a well-established and sensitive test in clinical settings, its application in preclinical research has been relatively infrequent. This report outlines the steps and criteria employed in performing stimulated SFEMG to quantify jitter and blocking in rodent models. This technique can be used in preclinical and clinical studies to gain insights into NMJ function in the context of health, aging, and disease.

Author Spotlight: Translational Applications of Stimulated SFEMG in Rodent Models

In this study, we demonstrate a refined single fiber electromyography (SFEMG) protocol to allow in vivo measurement of neuromuscular junction (NMJ) transmission in rodent models. A step-by-step approach to the SFEMG technique is described to allow quantification of NMJ transmission variability and failure in rat gastrocnemius muscle.

As the final connection between the nervous system and muscle, transmission at the neuromuscular junction (NMJ) is crucial for normal motor function. ...

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

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