Abstract
The ankle dorsiflexor muscles of rabbits
were denervated by ligation of the common peroneal nerv
1
Introduction
Functional Electrical Stimulation (FES) is
the use of electrical stimulation to restore movement and posture lost as a
result of neuromuscular injury. Although it has been studied extensively in
relation to paralysis brought about by upper motor neurone injury, much less is
known about its potential therapeutic use in cases where lower motor neurones
are also damaged. Such injuries result in severe denervation atrophy and
degeneration of the affected muscles. Could replacement of the lost neural
activity by electrical stimulation reverse any or all of these changes?
The time course of changes resulting from denervation
has been well characterised in the rat [1]. Some experimental work has been
done on the effect of stimulation on denervated muscles in this species [2].
Stimulation of a denervated muscle calls
for larger currents and longer pulses than those needed to elicit a response
from an innervated muscl
We have developed, in conjunction with the
Department of Biomedical Engineering & Physics,
Little data is available on the effects of
long-term denervation, with or without
2
Methods
Under general
anaesthesia and with full aseptic precautions, the common peroneal nerve of seven
male New Zealand White rabbits (2.5 – 3 kg) was avulsed and ligated, resulting
in the denervation of the ankle dorsiflexor muscles. A custom-made electronic
stimulator was placed in the peritoneal cavity and secured to the abdominal
wall. Two subcutaneous leads terminated in large (88 mm2) stainless
steel foil electrodes, which were secured to the proximal superficial
and deep distal surfaces of the tibialis anterior (TA) muscl
After a
period of 10 weeks of denervation four animals were lightly restrained and the
implanted stimulator was contacted via a radiofrequency link. The stimulation parameters
were defined and established via this radiofrequency link with software running on a
lap-top PC. The stimulators were set to deliver a daily pattern of stimulation
for 1 hour per day, which was continued for a period of 6 weeks.
The
stimulation pattern consisted of a series of 20 ms bipolar square pulses with
an amplitude of 4 mA, in bursts that were ON for 1 s and OFF for 2s.
Figure 1: Schematic
representation of the stimulation pattern used.
At the end of
the experiment the animals were anaesthetised for a terminal procedure in which
the physiological properties of the TA muscle were assessed.
The TA muscle
of the experimental (left) hind limb was exposed through a skin incision. The muscle
was partially freed from connective tissue attachments to the EDL and its distal
tendon was cut and fixed to miniature clamps, which facilitated attachment to a
servometer and force transducer. The tendons of all other muscles in the lower
limb were cut to remove possible influence of these muscles on the force
generated. This procedure was repeated on the innervated contralateral muscle
of the right hind limb (TAR), after attaching electrodes to the muscle and cutting
the common peroneal nerv
Muscle
mechanical properties were assessed using standard protocols. The force-frequency
relationship was determined. In the innervated TA muscle, frequencies from 5 to
50 Hz were tested, whereas in the contralateral innervated TA muscle
frequencies from 10 to 200 Hz were tested. (The upper frequency limit in the denervated
TA was set by the requirement for a stimulus pulse duration of 10 ms.) With the
optimized stimulation parameters (amplitude, pulse duration and frequency) the
relationship between force generated and velocity of contraction was
determined. Finally, a “Burke fatigue test” was conducted. This involved monitoring
the force while the muscle was stimulated with bursts of 330 ms duration,
repeated once a second for 15 min, with a stimulation frequency of 40 Hz and
optimal stimulation amplitude and pulse width.
At the end of
the physiological assessments the animals were humanely killed by an
intravenous overdose of sodium pentobarbiton
The sections
were studied by quantitative morphometry as follows. A transparent grid of 1 mm2
was placed over the section and the total cross-sectional area of the muscles
measured. This grid also provided the basis for a random selection of fields of
view that were subjected to morphological assessment, with a total of 10 fields
being analysed. The total number of fibres present within each field of view
was combined with the measurement of muscle cross-sectional area to provide an
estimate of the total fibre number within the muscles. An eye-piece grid of 100
squares was utilised to obtain a point count estimate of the area occupied by
the different components of muscle (muscle fibres, connective tissue, fat or
blood vessels or nerves).
3
Results
In the following account all figures are
quoted as mean ± SEM.
Denervation of the left TA muscle for a
period of 10 weeks resulted in a significant (p<0.001) decrease in muscle
mass to approximately 50% of the contralateral control (Den: 1.97±0.3 g vs. TAR: 3.91±1.97 g).
This decrease was mirrored in a decrease in cross-sectional area of the TA
mid-belly (38.5±1.6 mm2 vs. 86.2±2.7 mm2). The total fibre number within the muscle
remained constant (15111±365 vs.
15281±837).
Denervation resulted in a significant
decrease in maximal tetanic forc
Table 1:
Physiological parameters of the contralateral (TAR), denervated and denervated
& stimulated muscles. Significant changes from TAR are denoted * =
p<0.05 , ** = p<0.01, *** = p<0.001.
|
|
TAR |
Denervated |
Denervated & Stimulated |
|
Twitch F
(N.g-1) |
0.87±0.1 |
2.68±0.3 * |
3.45±0.3 *** |
|
Tetanic F (N.g-1) |
7.69±0.2 |
4.79±1.0 *** |
4.98±0.3 *** |
|
Twitch rise
time (ms) |
21±0.8 |
52±5.6 *** |
50±3.1 *** |
|
Twitch relax
time (ms) |
16±0.8 |
37±4.2 *** |
33±3.1 *** |
|
Vmax (mm.s-1) |
396±96 |
114±10 *** |
326±44 ** |
|
T60 (s) |
189±53 |
113±5 |
165±31 |
Denervated
muscles that were subjected to 6 weeks of stimulation failed to show
significant changes (Students t-test) in muscle mass (2.65±0.2g), muscle cross-sectional area (62.5±8.8mm2), fibre number (18236±885), or any measured physiological parameter relative to muscles
that were subjected to denervation alon
4
Discussion and Conclusions
The 50%
decrease in muscle mass we observed in the 10-week denervated muscle was
similar to that reported in other species [3]. The decrease in mass was
mirrored in a decrease in the cross-sectional area of the TA mid-belly.
Denervation
resulted in a slowing of contractile characteristics, the possible consequence
of selective type 2 atrophy, as observed by others and ourselves (results not
shown).
The
stimulation regime produced significant improvements in muscle morphology, yet no
corresponding improvements were seen in functional characteristics.
We are currently investigating whether a
more intensive stimulation regime would result in functional improvement,
particularly after denervation for longer periods.
References
[1]
[2]
Hennig R, Lomo T. Effects of
chronic stimulation on the size and speed of long-term denervated and
innervated rat fast and slow skeletal muscles. Acta Physiol Scand, 130:
115-131, 1987.
[3]
Kobayashi JSE, Mackinnon SE,
Wantanabe O, et al., The effect of duration of muscle denervation on
functional recovery in the rat model. Muscle Nerve 20: 858-866, 1997.
Acknowledgements
Both the implantable electronic stimulator
and the bench-top stimulator were designed and built by collaborating
colleagues in the Department of Biomedical Engineering & Physics, Medical University,
AKH, A-1090, Vienna, Austria.
The research was supported by European
Union Project ‘RISE’ (QLG5-CT-2001-02191).