Section 9-e
Analysis of Motor Unit Reinnervation in
Muscles of the Transplanted Hand
Marco Pozzo, Dario Farina
Introduction
Sensory and motor recovery in hand-transplanted patients is conditioned by nerve regeneration
[1]. Whereas functional recovery can be evaluated clinically [2], there is a need for tools allowing
direct assessment of muscle control in transplanted muscles at the level of their smallest
functional units, the motor units (MUs). These
should permit, for example, the ability to determine when MUs are innervated and whether
their control strategies and physiological properties are similar to those observed in normally
innervated muscles. These issues can be assessed
by techniques that allow extraction of the electrical activity from single MUs, such as intramuscular electromyography (EMG). However,
noninvasive methods detecting EMG signals on
the skin (surface EMG) should be preferred in
hand-transplanted subjects to minimise possible
damage to the allograft.
Recently, advanced EMG techniques (multichannel surface EMG [3]) have been advantageously applied to assess the reinnervation
process in intrinsic muscles of the transplanted
hand at the finest level of single MU activities.
Such methodology allows the detection of early
signs of reinnervation in intrinsic muscles of the
transplanted hand when only few MUs are reinnervated and the exerted force is too weak to be
perceived. In addition, after reinnervation, it
allows the investigation of MU physiological and
control properties and their functional recovery.
This chapter reviews the most recent findings
in the application of multichannel surface EMG
in the field of hand transplantation. Due to the
innovative nature of this methodology, most of
the findings illustrated in this chapter will refer
to the postoperative follow-up of one patient
[3–5]. The technique has also been applied to a
second recipient, of whom preliminary results
will be shown in this chapter.
The Multichannel Surface EMG
Technique
Muscle fibres are activated by the central nervous system through electric signals transmitted
by the motoneurons. A motoneuron innervates a
group of muscle fibres (in a range from few tens
to several hundreds) that constitute an MU, the
smallest functional unit of the muscle, which is
controlled independently. MU activation by the
central nervous system can be assessed by the
detection of electrical signals (MU action potentials) generated before their contraction [6].
Surface EMG signals reflect the electrical activity of the active MUs in a muscle. When an electrical signal reaches the neuromuscular junction
through the axon branches, two action potentials
are generated at the end-plate region (innervation zone) and travel by active propagation
towards the tendon endings at a speed (termed
conduction velocity) related to MU membrane
and contractile properties [7] and eventually
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fade at each tendon. Intracellular action potentials generated in the muscle fibres are the
sources of the surface EMG signal detected over
the skin.
Classic techniques for the detection of surface EMG signals consist of pairs of electrodes
spaced at 20–30 mm and aligned with musclefibre orientation. A signal, which is the difference of the electric potentials detected by the
two electrodes, is recorded [single differential
(SD) or bipolar recording). The surface EMG
technique is particularly attractive in the conditions of slow reinnervation processes since in
these cases, only a few MUs are active. Despite
the lower spatial resolution of the recording with
respect to the intramuscular technique, it is possible to separate the interference EMG signal
into its constituent action potentials generated
by the active MUs.
Surface recording has many advantages over
intramuscular detection, avoiding risks of infections and discomfort issues (which are of particular importance in this specific application),
despite the fact that it can provide only global
indications on muscle activity. More advanced
methods for surface EMG signal recording have
been proposed [8] with the aim of investigating
single MU anatomical, action potential propagation and control properties. These methods
make use of linear electrode arrays, i.e. a number
of equally spaces electrodes placed parallel to
fibre orientation, in which each consecutive electrode pair originates an SD EMG signal (Fig.
1c). Detection of such multichannel EMG signals
allows identification of the MU innervation zone
location, tendon placement, fibre length, conduction velocity and, in some conditions, discharge patterns [8–11]. Figure 1 shows examples
of surface EMG signals recorded by a linear electrode array (16 dot-shaped electrodes, 2.5-mm
interelectrode distance), from the abductor digiti minimi muscle of a healthy male subject during a linearly increasing force contraction from
0% to 100% of the maximal voluntary contraction (MVC). During the ramp contraction, the
EMG signal amplitude increases (Fig. 1a) as a
consequence of MU recruitment and the
increase of MU discharge rate [i.e. mean number
of MU action potentials generated per second
and measured in pulses per second (pps)]. A
large number of MU action potentials are present when the force level increases.
In a (re)innervated hand muscle, the location
of the innervation zone of active MUs can be
assessed by visual analysis of the surface EMG
signals and corresponds to the point of inversion
of propagation of the MU action potentials [9],
as shown in the example from a healthy subject
(Fig. 1d) When EMG activity in the allograft is
evident, single MU action potentials can be
extracted from the signal by means of dedicated
signal processing algorithms, which classify MU
action potentials based on their shape as belonging to different MUs. It is then possible to identify with precision when a new MU is reinnervated and to analyse the membrane and control
properties of each MU individually.
The instantaneous discharge rate of each MU
can be calculated as the inverse of the time interval between consecutive discharges. This parameter gives indication on the capability of the
recipient to modulate the motor control of the
reinnervated MUs in specific tests. MU conduction velocity can be estimated from the highest
available number of propagating signals with
methods described in the literature [12]. Its
value can give an insight into membrane and
physiological properties (such as fatigability) of
the innervated MUs.
Procedures for Follow-Up Assessment of Reinnervation
Assessment of early signs of reinnervation in the
transplanted hand is performed by periodical
EMG recording sessions, starting a few months
postoperatively, in which evidence of electrical
activity from intrinsic muscles is evaluated. The
first case analysed with this methodology was a
35-year-old male recipient who had lost his right
dominant hand at the age of t13. Recordings of
EMG activity started 7 months postoperatively,
followed by a second evaluation at 11 months
and then monthly thereafter, until reaching 10
sessions. An additional session was then performed 4 months after the 10th session. The sec-
Analysis of Motor Unit Reinnervation in Muscles of the Transplanted Hand
309
a
b
c
d
Fig. 1a-d. Multichannel surface electromyography (EMG) signals acquired from the abductor digiti minimi muscle of a healthy
subject during a 30-s increasing force ramp contraction from 0% to 100% of the maximal voluntary contraction (MVC). A 16channel, 2.5-mm interelectrode distance array with silver dot electrodes was used to acquire EMG signals. a Time course of one
EMG channel, showing an evident increase in the global amplitude. b One-second epochs of EMG signals extracted from the
recording at the beginning, middle and end of the contraction.Note the increase in the firing rate of active motor units (MU) and
the progressive recruitment of larger MUs as the force demand increases.c The electrode array used to acquire multichannel surface EMG signals.The silver dot electrodes are equally spaced by an interelectrode distance of 2.5 mm. During EMG acquisitions,
the array is positioned parallel to the muscle fibre direction and held in place by applying a gentle pressure on the skin.d Sample
portion of multichannel surface EMG signals acquired from the abductor digiti minimi muscle from a healthy subject (16 channels, 2.5-mm interelectrode distance array, 10% MVC) showing its features. Each MU, when active, produces a train of MU action
potentials traveling from the innervation zone (IZ) towards the tendons, originating typical V-shaped patterns. The channel
where sign reversal is observed corresponds to the location of the innervation zone.The time delay of potentials travelling under
consecutive electrodes is related to the MU conduction velocity, the normal value of which is approximately in the range 3–5 m/s
ond recipient was a 32-year-old male who lost
his right dominant hand 7 years earlier. In this
case, EMG recording sessions started at month 3
postoperatively, and 3 additional sessions were
performed at months 6, 7 and 13. Muscles that
can be investigated with this method are the
abductor digiti minimi, abductor pollicis brevis,
opponens pollicis, first dorsal interosseous and
first lumbricalis. Indeed, these are sufficient to
provide an overview of the reinnervation status
in a transplanted hand.
For the EMG assessment, the skin overlying
the muscle to be investigated is slightly abraded
with abrasive paste to improve the quality of the
skin–electrode contact. The electrode array is
held in place by an operator who explains to the
subject the specific movement to perform to activate the muscle and provides an appropriate
counterresistance. In case of presence of EMG
signals, the final location of the array is determined by visual inspection of the signals detected while the subject is performing short test con-
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tractions. The best electrode location is defined
as that corresponding to the propagation of the
MU action potentials along the array with minimal shape changes. In case of absence of EMG
activity, the array is placed along the muscle fibre
direction, as estimated by muscle palpation.
Surface EMG signals are amplified by a multichannel surface EMG displayed in real-time on
a monitor and stored on a computer for further
processing and analysis [3]. For each muscle, the
subject is asked to perform a 60-s contraction at
maximal level and is verbally encouraged to
increase the force level. In case no EMG activity
is observed, the muscle is considered not innervated, and no other measures on the muscle are
performed in the same experimental session. In
case clear MU action potentials are identified,
the subject is asked to perform three additional
contractions, increasing linearly the muscle
activity from zero to the maximum (subjective
regulation of force). When a single MU action
potential train is identified, the subject is also
provided with visual feedback that displays the
MU instantaneous discharge frequency on a
visual analogue scale. Such feedback allows the
subject to linearly increase in discharge rate
from a minimum to the maximum. This ramp
contraction serves to test the MU control strategies in a simple force-production task.
Electrophysiological Evidence of
Motor Unit Reinnervation
In the first recipient, the first clear MU potential
train appeared from the abductor digiti minimi
muscle (Fig. 2a) 11 months after the allograft
procedure. Analysis of EMG signals allowed
determination of the point in which the axon
connected to the muscle fibres (Fig. 2f ).
Observed discharge rates were within physiological values (with a minimum of 8–10 pps and a
maximum of 35–40 pps) [13, 14], except for
occasional multiple discharges very close to each
other (reaching instantaneous firing rates up to
100 pps). These discharges resembled the double discharges observed both in healthy [15] and
pathological subjects [16], but in the investigated subject, more than two discharges often
appeared very close to each other. The estimated
conduction velocity was within physiological
values, in the range 3–4.5 m/s, and it depended
on the discharge rate, as shown below. After 13
months, a second MU appeared during maximal
contractions of the abductor digiti minimi muscle. Surface potentials of this unit presented significantly smaller amplitudes than those of the
first observed MU, indicating either a deeper or
a smaller MU. After 12 months from transplant,
abductor and opponens pollicis muscles began
to show single MU surface EMG activity (Fig.
2b, c). A clear MU action potential train was
observed in the opponens pollicis muscle while,
at the time in which reinnervation was first
observed, at least 3 MUs were detected from the
abductor pollicis muscle. Also in these muscles,
instantaneous discharge rates were within physiological values. After 15 months, the first dorsal
interosseous muscle showed the first active MU
(Fig. 2d), made manifest by a train of action
potentials. Activity from the first lumbricalis was
first detected 24 months postoperatively
although the amplitude of the MU action potential train was lower than in the other muscles.
For the abductor digiti minimi, abductor pollicis, and opponens pollicis muscles, from the
EMG recordings it was possible to clearly identify the MU innervation zones, which could be
marked over the skin (Fig. 2f).
In the second recipient, the smaller number
of evaluation sessions did not allow determination of the reinnervation sequence with the same
precision. However, in this case, the reinnervation process was faster, with the first clear MU
action potentials detected on the abductor digiti
minimi in the session at month 7 postoperatively. By the fourth measurement session (month 13
postoperatively), the opponent and abductor
pollicis and first lumbricalis also showed MU
action potential trains. In the case of the opponent pollicis, at least two MUs could be identified while no activity was observed in the first
dorsal interosseous in any of the sessions. In all
reinnervated muscles, it was possible to observe
signal propagation (Fig. 3).
Analysis of Motor Unit Reinnervation in Muscles of the Transplanted Hand
a
b
c
d
e
f
311
Fig. 2a-f. Multichannel surface electromyography (EMG) signals acquired from intrinsic muscles of the transplanted hand of first
recipient during attempted voluntary contractions against the resistance of the operator. For each muscle, the date when voluntary EMG activity was observed for the first time is indicated. Only the channels with high enough signal quality were plotted in
each case.A 16-channel, 2.5-mm interelectrode distance array with silver dot electrodes (as shown in Fig. 1c) was used to record
EMG signals. Note the different amplitude scale for each graph. The investigated muscles were: (a) abductor digiti minimi, (b)
abductor pollicis brevis,(c) opponens pollicis,(d) first dorsal interosseous,(e) first lumbricalis.f Position of the array for the investigated muscles (except for the first dorsal interosseous).For each muscle, the two crosses (+) represent the location of electrodes
1 and 16 of the array, and the dashed line (- - -) indicates array direction. For the muscles in which signal quality and number of
propagating channels was high enough,the estimated position of the innervation zone ( IZ) is also marked.From [3],used with
permission
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M. Pozzo , D. Farina
a
b
c
d
Fig. 3a-d. Multichannel surface electromyography (EMG) signals acquired from intrinsic muscles of the transplanted hand of
second recipient during attempted voluntary contractions against the resistance of the operator. Plots refer to EMG recordings
obtained at month 13 postoperative from: (a) abductor digiti minimi, (b) abductor pollicis brevis, (c) opponens pollicis, (d) first
lumbricalis. No activity was detected on the first dorsal interosseous. A 16-channel, 2.5-mm interelectrode distance array with
silver dot electrodes (as shown in Fig. 1c) was used to detect EMG signals. Signals are depicted in arbitrary units (AU), with different vertical scales for each muscle for best visualization. Only channels with good signal quality and clear propagation are
shown
Figure 4 shows a 1-s segment of surface EMG
signals detected from the abductor digiti minimi
of the first recipient during a 60-s maximal voluntary contraction. Fluctuation of discharge rate
is evident, as is the occasional presence of multiple discharges at high instantaneous rate. In all
the 60-s contractions sustained at the maximal
level, the mean discharge rate decreased on average, probably reflecting central phenomena of
fatigue, despite the verbal encouragement given
to the subject to keep it at the initial level. Figure
5 shows a ramp contraction of the abductor digiti minimi performed by the first subject with the
feedback on discharge rate. The subject was able
to approximately increase the frequency of activation of the MU linearly in time from about 10
up to approximately 40 pps. The occasional high
discharge frequency values can be observed from
the plot of the instantaneous discharge rate.
Interestingly, conduction velocity shows high
correlation with instantaneous discharge rate, as
it was also observed in normal subjects [17],
indicating that membrane properties depend on
the time elapsed from the previous discharge.
Figure 4e shows the action potentials classified as
belonging to the MU under study. The subject
was able to perform this simple ramp motor control task (constituted by the linear increase of
single MU discharge rate) since the beginning of
the reinnervation and with all muscles from
which it was possible to extract single MU activities. The minimum discharge rate that could be
Analysis of Motor Unit Reinnervation in Muscles of the Transplanted Hand
313
Fig. 4. Multichannel surface electromyography (EMG) signals acquired from the abductor digiti minimi muscle of the transplanted hand of first recipient during attempted maximum voluntary contraction (MVC).The subject was asked to exert the maximum
possible force against the resistance of the operator and keep it for 60 s; the subject was verbally encouraged during the contraction, but no feedback was given to him. A 16channel, 2.5-mm interelectrode distance array with silver dot electrodes (as
shown in Fig. 1c) was used to acquire EMG signals. One epoch of EMG signals, one second long, at the beginning of the contraction (12–13 s) is shown. Despite the fact that exerted force was almost not perceivable by the operator, the effort of performing a maximal contraction reflects in the high firing rate of the only detected motor unit. Occasional bursts of multiplets (shaded area) with high firing rate (approaching in this case 50 pps) can be observed
sustained constantly was approximately 8–10
pps in all conditions, and the maximum firing
rate, sustained for at least 2 s, was never higher
than 40 pps, which is similar to those observed
in normal subjects. Similar phenomena were
observed in the second recipient.
Physiological and Clinical
Implications
Analysis of MU properties opens a window on
the understanding of central control strategies
and peripheral status of the neuromuscular sys-
tem. Using the technique described in this chapter, the activation of single MUs from intrinsic
hand muscles can be followed after the transplant operation. The electrical activity of such
muscles shows that small forces perceived by the
therapist in the transplanted hand are not only
due to synergic efforts performed by extrinsic
muscles.
Anatomical information about the muscle
can be obtained by localisation of innervation
zones of the detected MUs. In addition, physiological information can result from the analysis
of both the discharge pattern and conduction
velocity of single MUs. Results from the first two
recipients analysed showed that the discharge
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M. Pozzo , D. Farina
a
b
e
c
d
Fig. 5a-e. Single motor unit (MU) parameters of surface electromyography (EMG) signals acquired from the abductor digiti minimi muscle of the transplanted hand of the first recipient during a 60-s voluntary ramp contraction.The subject was given realtime feedback of the instantaneous firing rate of its active MU and was instructed to follow a target, varying at small steps from
the minimum to the maximum firing rate that he could exert. a Time course of conduction velocity (CV) of the active MU () and
its interpolating curve (- - -). Note the high and instantaneous correlation between the MU conduction velocity and firing rate
(b). b Time course of the instantaneous firing rate of the active MU () and its interpolating curve (- - -). Note that despite the
fluctuations the subject was able to increase the MU firing rate as requested. c Time course of one EMG channel. Note the constant amplitude with respect to Fig. 1a due to the only active MU contributing to the signal.d Epochs of EMG signals (three channels shown), 1 s long, extracted from the signal at the beginning (11.0–12.0 s), middle (30.0–31.0 s) and end (52.0–53.0 s)
of the ramp contraction. Note the increase of the firing rate. e All the MU action potentials extracted from the signal (dark grey
lines).All propagating channels used to compute conduction velocity are shown;the average MU action potential is shown superimposed (black lines). Note the similarity of all MU action potentials with their average, which confirms that they all belong to
the same MU.The jitter in the shape is due to fluctuations of the CV, which are evident in a, as described in the text
rates achieved by the patients were within the
range of physiological values (8–40 pps). Stable
discharge rates were never below 8 pps, which is
a finding common to a number of muscles in
normal conditions [14]. Occasionally, high
instantaneous discharge rates (up to 100 pps)
were recorded (Fig. 4). They corresponded to
discharges very close to each other, which could
resemble “doublets” [15] identified in normal
subjects but that in this case involved usually
more than two discharges (“multiplets”).
Multiple discharges may reflect an attempt of the
central nervous system to exert an increasing
force when few MUs are available. In addition,
the subjects were able, with limitations but with
increased skill over the sessions, to voluntarly
control the innervated MUs by increasing their
discharge rate when requested. For the muscles
in which conduction velocity could be estimated,
its values were within normal physiological
ranges and correlated to MU discharge rate, as it
has been observed in normal subjects.
Analysis of Motor Unit Reinnervation in Muscles of the Transplanted Hand
In conclusion, advanced noninvasive EMG
techniques can monitor the reinnervation of single MUs in transplanted hands. The location in
the muscle in which the neuromuscular junctions are restored can be detected, and the membrane and control properties of the innervated
MUs can be investigated and compared with
315
those of normal subjects. Selective assessment of
intrinsic muscles in the transplanted hand is
thus feasible even at the lowest functional level,
the MU. This assessment provides important
information from clinical and basic physiological perspectives and discloses new research areas
in limb transplants and motor control studies.
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