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, 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.
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).
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.
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') 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-α 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.
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
Figure 3: Experimental setup. Classical experimental setup to record electromyographic (EMG) and torque signals.
2. Testing Procedures at Rest
3. Testing Procedures During Voluntary Contraction
4. Data Analysis
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. Please click here to view a larger version of this figure.
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.
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’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.
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. Please click here to view a larger version of this figure.
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.
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’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’ 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 activation61–63. 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 level65–67.
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 maximally69–71. 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’s disease79. Neuromuscular plasticity can also be assessed in pathological populations during therapy/retraining program80.
The authors have nothing to disclose.
The authors have no acknowledgements.
Biodex dynamometer | Biodex Medical System Inc., New York, USA | www.biodex.com | |
MP150 Data Acquisition System | Biopac Systems Inc., Goleta, USA | ||
Acknowledge 4.1.0 software | Biopac Systems Inc., Goleta, USA | www.biopac.com | |
DS7A constant current high voltage stimulator | Digitimer, Hertfordshire, UK | www.digitimer.com | |
Silver chloride surface electrodes | Control Graphique Medical, Brie-Comte-Robert, France | ||
Computer | |||
1 Cable for connecting the Biodex to the MP150 | |||
1 Cable for connecting the Digitimer to the MP150 | |||
1 Cable for connecting the MP150 to the computer |