NEURODIAGNOSTICS

Clinical Note

Repetitive Nerve Stimulation

– Exercise Testing

www.natus.com
By Antonine MULOT
International Clinical Specialist, Natus Neuro

Abbreviations

EMG: electromyography
RNS: repetitive nerve stimulation
Ach: Acetylcholine
NMJ: neuromuscular junction
CMAP: compound motor action potential
MG: Myasthenia Gravis
LEMS: Lambert-Eaton Myasthenic Syndrome

Introduction

Some of the most common neuromuscular junction disorders are Myasthenia Gravis, Lambert-Eaton
Syndrome, and Symptomatic Botulism. Such pathologies can be diagnosed by an array of tests that are more
or less specific, for example Single Fiber EMG and Repetitive Nerve Stimulation. Single Fiber EMG testing
requires patient cooperation, and may be very time consuming, while Repetitive Nerve Stimulation/Exercise
Testing can be more easily performed in non-cooperative patients and with less time available. The following
paper reviews the technique lying behind RNS and Exercise Testing.

Neurophysiological and pathophysiological background

The neuromuscular junction is the structure that links nerve terminals to muscle fibers. A chemical
transmitter, called Acetylcholine (Ach), serves as a mediator to overcome the electrical mismatch between
the presynaptic fine nerve fibers and postsynaptic large muscle fibers for proper electrical signal
transmission.

However, Ach is not directly active. It is synthetized in the nerve terminal and stored in vesicles. Those
vesicles are partitioned in several compartments, the smallest called the “mobilization store”, which is the
first one used for release in case of need.

When a nerve impulse reaches the nerve terminal, it opens voltage-gated calcium channels. Calcium ions
enter the neuron, forcing the vesicles to fuse with the nerve membrane, therefore releasing Ach into the
synaptic cleft.

On the other side of the cleft are located the muscle membrane folds, containing Ach receptors. The binding
of Ach opens the receptor for a few milliseconds, ensuring sodium can create local depolarization at the
muscle membrane. This initiates the process of muscle contraction.

Each release of vesicles is called a quanta. Upon nerve stimulation, up to hundred quanta can be released. If
the depolarization at the muscle membrane reaches a sufficient level, muscle contraction can occur. In
normal subjects, excess of Ach is released in the synaptic cleft, ensuring that depolarization will be sufficient.
This is called the safety factor. If the safety factor is diminished, neuromuscular transmission may be
blocked.

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Two main types of NMJ disorders can be distinguished: postsynaptic abnormality (present in Myasthenia
Gravis) and presynaptic abnormality (present in Lambert-Eaton syndrome).

In Myasthenia Gravis, Ach receptors are disturbed (see Figure 1).

In Lambert-Eaton syndrome, calcium channels disturbance at the nerve terminal is responsible for the
pathology. The presynaptic active zone is reduced in terms of area, thus one quanta can be efficiently
released, but the total number of released quanta is severely decreased when compared to healthy patients.
The global effect is thus a limited muscle membrane depolarization as a result of nerve stimulation.

Figure 1 : transmission through the neuromuscular junction. In the middle part, a normal neuromuscular
junction process, Ach is released into the synaptic cleft and binds to Ach receptors, enabling muscle
contraction. On the left side, the location of disturbance for botulin toxin. On the right side, the agents
responsible for MG and Lambert-Eaton diseases. In MG, antigenic agents are blocking Ach receptors after the
synaptic cleft. In LEMS, antibodies disturb normal operation of calcium channels.

This note focuses mainly on MG and LEMS findings, however more pathologies may be assessed in the NMJ
dysfunction using RNS and Exercise Testing. The following table, provided by the AANEM, showcases the
diversity of pathologies involving the neuromuscular junction.

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 Presynaptic Synaptic Postsynaptic
Lambert-Eaton Myasthenic Organophosphates Myasthenia gravis
Syndrome Edrophonium Neonatal myasthenia
Botulism Neostigmine Congenital myasthenia
4-aminopyridine Congenital end-plate AChE Succinylcholine
Steroids deficiency D-penicillamine
Magnesium Curare
Black Widow venom Classic slow-channel syndrome
Congenital myasthenic syndrome
Aminoglycosides
Tetanus
Tick paralysis

Technique

Repetitive nerve stimulation relies on a train of stimulations (at 3-4 Hz) on a motor nerve with recording on a
muscle innervated by this nerve. Supramaximal stimulation is required for such testing. CMAP is recorded for
each stimulus and amplitude changes are assessed. The amplitude change is referred to as decrement.

Additional exercise can be performed, and RNS then transforms into Exercise Testing.

The exercise can be constituted by a rapid train of electrical stimulations or by voluntary muscle contractions
by the patient. Both of these tasks induce facilitation period.

Rapid stimulation relies on a 40-50 Hz stimulation during 2 seconds. The rapid stimulation increases the
intracellular calcium concentration at the nerve terminal and mobilizes Ach stored in secondary vesicles. This
action increases the release of Ach in the synaptic cleft for 50 to 100 seconds.

A vigorous contraction of the muscle can replace this technique in a painless way, creating a 20-30 Hz
stimulation. This will have the same effect as the rapid repetitive stimulation, however does require a
cooperative patient.

After the exercise period, additional RNS are repeated after some period of rest (up to 5 minutes). The
decrement shall be similar to the first RNS after sufficient rest in case of a normal neuromuscular junction.

Electrodes

Recording electrodes are preferably pre-gelled electrodes. The patient’s skin shall be cleaned beforehand
using Nuprep paste. This ensures a better impedance, therefore less artefacts during recording.

Recommended electrodes for stimulation are also adhesive electrodes. Patient’s skin cleaning is identical to
what described above.

Below are examples of suitable electrodes and gel for RNS test:

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 122-736100 Nuprep Skin Prep gel

019-400400 Disposable 4-disc electrode set with lead

019-400500 Disposable ground plate electrode with lead

Patient preparation

If possible, withdraw anticholinesterase medication minimum 8 hours prior to testing.

Install patient in a comfortable position with supported limb to minimize muscle movement and muscle
contraction. Alternatively, it is possible to fix the hand to the arm of the chair or to a piece of wood. Hand
clamp can also be used as an alternative.

Ensure that the limbs are warm, at least 34-36 degrees Celsius. If needed, warm them up prior to test start.

Recording electrodes are placed on the muscle of interest: active electrode over the muscle belly and
reference electrode over the distal tendon.

The ground electrode does not require any specific position, however ensure that the ground does not
overlap any other electrode.

Figure 2 : electrode placement for ulnar nerve testing. Active recording electrode is placed on the belly of the
ADM muscle, reference electrode over the distal tendon. Hand is secured with a strap.

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System setup

Stimulator settings:

Stimulator settings are equivalent to any motor test stimulation.

Step 1
Supramaximal response identification phase
Stim duration : 0.2 ms
Monophasic stimulation

Step 2
Train of stimuli
Stim duration : 0.2 ms
Monophasic stimulation
5-10 pulses
Frequency rate : 3 Hz

Amplifier filters

Amplifier settings are equivalent to any motor nerve conduction study.

High pass: 20 Hz
Low pass: 2-5 Hz
Sensitivity: 2-5 mV/div
Sweep speed: 2 ms/div

Data acquisition and analysis – exercise testing

1st step: setup mode to record motor supramaximal response

Use manual single stimulation and record supramaximal response. Measure negative amplitude and
negative area. Add 25% to stimulus intensity before going to next step.

2nd step: baseline train stimuli – repetitive nerve stimulation

Run a train of stimuli with 10 stimuli at 3-4 Hz and record every single response. User wants to compare
amplitude decrease throughout the train of stimulation. Generally potentials 1 and 4 are compared,
potentials 1 and 10 may also be compared. Such comparison is referred to as decrement.

3rd step: patient exercise

For a cooperative patient, perform maximum voluntary contraction for minimum 10 seconds, usually 20
seconds. For difficult / non-cooperative patients, tetanic stimulation may be used. In this case, a train of 100
stimuli at 50 Hz may be delivered.

4th step: post exercise trains

One RNS train shall be performed immediately after the exercise, then extra RNS trains shall be performed
each minute up to 5 minutes post-exercise.

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 Figure 3 : example of exercise testing on EMG software. The top left window displays the supramaximal
response. Top right window displays responses to train stimulations. The middle part showcases the testing
workflow in blocks, with train of stimulation prior to the exercise (named “Activation” in the sw), exercise
instructions and series of stimulation trains after the exercise. Each block contains amplitude and area of the
first response and the amplitude histogram over the train. Additionally, on the bottom part, plots can
showcase the evolution of decrement over the duration of the test.

Interpretation considerations

The reason behind the comparison between peak 1 and 4 is that the primary store of vesicles is mainly
depleted near the 4th stimulus. This is the cause of increasing blocking at the neuromuscular junction. After
that, secondary stores are involved and the Ach contained in a larger number of vesicles is released again
and the amplitude of responses increases proportionally. This can also explain the parabolic shape of train
amplitudes in train/histogram visualization.

Figure 4 : responses to train stimulation: the amplitude of the response decreases up to the 4th stimulation,
then slightly increases again, giving the parabolic shape.

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A decrement up to 8% is considered acceptable for normal patients.

In the case of MG, a decrement of the CMAP amplitude of 10% or more shall be observed. Muscles that are
clinically weak shall be examined first. A brief exercise (around 10 seconds) usually induces a reduction of
the decrement value right after the exercise by post exercise facilitation.

Right after the exercise, the area under the curve and the peak to peak amplitude of the motor response
may be larger, this effect is called postactivation potentiation. It results from an increased number of quanta
released leading to the activation of more muscle fibers (facilitation) or from an synchronization of action
potentials generation at the level of the muscle membrane (pseudofacilitation).
In case of NMJ pathology, this excess of ACh release may improve synaptic transmission briefly. The
assessment of the motor response area under the curve before and after exercise can distinguish
postactivation potentiation from pseudofacilitation. When pseudofaciliation is involved, the amplitude of
the response increases after exercises but the area under the curve of the response remains the same.
A few minutes after exercise, the amplitude of the motor response in a myasthenic muscle can be smaller
and decrement greater than at rest. This is called postactivation exhaustion. In mild myasthenia,
postactivation exhaustion can be the only abnormality showing up. Therefore, it is important to keep
running stimulation trains up to 5 minutes after the exercise step.

Figure 5 : exercise testing in MG. Prior to the exercise, the amplitude decrement between stimulation 1 and 4
is 51%. Decrement is then reduced right after the exercise to 18%.

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In the case of LEMS, one shall observe an increase of the CMAP amplitude after brief exercise or rapid
repetitive stimulation, usually over 200% increase. This increase results from calcium facilitation of
transmitter released during the exercise. This increase, together with the low initial amplitude at rest, is a
typical finding in LEMS patients.

Figure 6: Exercise testing in LEMS. The CMAP amplitude prior to exercise is 5.57 mV and increases after the
exercise to 10 mV. Moreover, the initial amplitude decrement between stimulation 1 and 4 reaches 41%,
exceeding 8% limit considered acceptable for normal patients.

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To better recognize LEMS, an additional rapid train of stimulation (from 20 to 50 Hz stimulation frequency)
for several seconds (between 2 and 6 seconds) may also be used and responses to each stimulus recorded.
After an initial decrease in amplitude, the amplitude increase between the first stimulus and the last
stimulus shall exceed 100%.

Figure 7: rapid train stimulation in LEMS. Ulnar nerve 50 Hz stimulation with recording over the hypothenar
muscle. Top trace displays the first 10 responses, bottom trace displays the amplitude change over the 5
seconds stimulation. The increment between the first and the last stimulus reached 233%, typical of a
presynaptic NMJ disorder.

In botulism cases, observations are similar to Myasthenia Gravis cases for initial train. However, brief
exercise will increase CMAP amplitudes, usually over 200%.

The shape of the train of CMAP responses can provide additional information about diagnosis. As
Myasthenia Gravis provides a parabolic shape, Lambert-Eaton syndrome creates a progressive decline of
amplitude rather than a parabolic shape.

User should pay attention to shape changes during RNS. Irregular changes between consecutive stimuli can
result from patient movement or electrode displacement. In such case, placement of the electrodes shall be
checked and a new RNS shall be performed.

Non optimal skin temperature should be avoided, as low skin temperature lowers the decrement value. If
needed, some warm water may be poured on the patient limb for a few seconds before starting the test.

Conclusion
In the assessment of NMJ dysfunction, Single Fiber EMG and RNS testing may be used for diagnosis. RNS
testing is easy to perform as long as the patient’s limb is stabilized. As observation of decrement is a typical
finding in NMJ abnormality, the addition of Exercise Testing can differentiate between presynaptic and
postsynaptic disorders, by observing changes in decrement value and CMAP amplitude prior and after the
exercise. The addition of area under the curve assessment can differentiate between two processes involved
in the decrement repair upon exercise in the case of Myasthenia Gravis.

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References

Electrophysiological study in neuromuscular junction disorders, Ajith Cherian, Neeraj N. Baheti, Thomas Iype,
Ann Indian Acad Neurol, 2013 Jan-Mar, 16(1): 34-41.

Electrodiagnostic approach to neuromuscular junction testing, Vern C. Juel, the American academy of
Neurology Institute, 2017

American academy of Physical Medicine and Rehabilitation website

Current status on electrodiagnostic standards and guidelines in neuromuscular disorders, A. Fuglsang-

Frederiksen, K. Pugdahl, Clinical Neurophysiology 122 (2011) 440-455

China guidelines for the diagnosis and treatment of myasthenia gravis, Zhu-Yi Li, Neuroimmunol
Neuroinflammation, volume 3, issue 1, January 20, 2016

Lambert-Eaton myasthenic syndrome: clinical diagnosis, immune-mediated mechanisms and update of

therapies, Sanders DB, Ann Neurol, 37:Suppl 1,S63, 1995

Aminoff’s Electrodiagnosis in Clinical Neurology, 6th edition, Michael J. Aminoff

Electromyography and neuromuscular disorders, clinical-electrophysiologic correlations, 3rd edition, David C.

Prestin, Barbara E. Shapiro

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