JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2012, 63, 3, 285-291
www.jpp.krakow.pl
J. WOJTKIEWICZ1,10, I.M. KOWALSKI2, Z. KMIEC3,4, R. CRAYTON5, I. BABINSKA6, M. BLADOWSKI7,
J. SZAREK6, W. KIEBZAK8, M. MAJEWSKI7, M. BARCZEWSKA1, W. GRZEGORZEWSKI9, W. KLOC1
THE EFFECT OF LATERAL ELECTRICAL SURFACE STIMULATION (LESS)
ON MOTOR END-PLATES IN AN ANIMAL MODEL OF EXPERIMENTAL SCOLIOSIS
1Department of Neurology and Neurosurgery, Division of Neurosurgery, University of Warmia and Mazury, Olsztyn, Poland;
Department of Rehabilitation, University of Warmia and Mazury, Olsztyn, Poland; 3Department of Human Histology and Embryology,
Faculty of Medicine Science, University of Warmia and Mazury, Olsztyn, Poland; 4Department of Histology, Medical University of
Gdansk, Gdansk, Poland; 5Department and Clinic of Urology, Faculty of Medical Sciences, Medical University of Warsaw, Warsaw,
Poland; 6Department of Pathophysiology, Forensic Veterinary Medicine and Administration, Faculty of Veterinary Medicine, University
of Warmia and Mazury, Olsztyn, Poland; 7Department of Human Physiology, Faculty of Medicine Sciences, University of Warmia and
Mazury, Olsztyn, Poland; 8Department of Physiology, Faculty of Health Science, University of Kielce, Kielce, Poland; 9Department of
Pharmacology and Toxicology, Faculty of Medicine Sciences, University of Warmia and Mazury, Olsztyn, Poland; 10Stem Cell
Research Labolatory, University of Warmia and Mazury, Olsztyn, Poland
2
The treatment of idiopathic scoliosis is challenging because of its diverse etiology, age of onset, and long duration of
intensive treatment. We examined the effect of lateral electrical surface stimulation (LESS) in an animal model of
experimental scoliosis (ES) assessing the number of motor end-plates (MEPs) as a study end-point. The control group
(n=5) was adapted to the experimental apparatus without stimulation, whereas ES was induced in rabbits by one-sided
LESS of the longissimus dorsi muscle (LDM) for a duration of 2 months. The ES group (n=5) were subjected to a shortterm corrective electrostimulation applied at the contralateral side of the spine compared to the previous LESS
stimulation for 2 h daily for 3 (n=5) or 6 months (n=5). Another group of ES rabbits was subjected to a long-term
corrective electrostimulation applied for 9 h daily for 3 (n=5) or 6 months (n=5). LESS applied for 2 months (ES),
significantly increased the number of MEPs in LDM. The short-term corrective electrostimulation for 3 months resulted
in an increased number of MEPs. However, a decrease was observed in the animals treated for 6 months. The long-term
corrective electrostimulation for 3 months did not change the density of MEPs in the LDM, but for 6 months the number
of MEPs in the LMD significantly decreased by ES and control groups. Thus, the results of the present study clearly
show that the short-term LESS is able to influence both the number of MEPs and the effectiveness of muscle correctional
adaptation in a more efficient and harmless manner than the long-term procedure.
K e y w o r d s : electrostimulation, lateral electrical surface stimulation, longissimus dorsi muscle, motor end-plates, scoliosis
INTRODUCTION
In Poland the occurrence of scoliosis in children and
adolescents ranges from 2% to 14% (1, 2), whereas Europewide, it affects 2-3% of the population (1). Moreover, scoliosis
accounts for 80-90% of all spinal deformities (3, 4).
The preferable treatment of scoliosis should target the
causative mechanisms involved in the development of this
condition. Previous studies have shown that the occurrence of
degenerative and atrophic lesions in both the muscle and
connective tissues which stabilize the spine, constitute changes
that are secondary to disorders of neuromuscular junctions (5-7).
Effective neuro-rehabilitation relies on the plastic capability
of neurons in the brain and motor units (7). Permanent posture
memory and learned behavior are both based on a long-term
potentiation. Hebb`s repetition effect states that the effective
stimulation of postsynaptic neurons by presynaptic neurons
strengthens the connections between them (8). The plasticity of
motor units on paravertebral muscles enables an active
modulation of the vertebral column shape thereby controlling the
posture (9). Previous studies have also indicated that electro
stimulation is not able to prevent the progression of scoliosis, thus
it cannot be employed as the only method of treatment (10, 11).
Non-invasive treatment by lateral electrical surface
stimulation (LESS) stimulates the intradermal proprioreceptors
which results in tetanous contractions of the underlying skeletal
muscles. The repeated application of stimulations increases the
tension of epaxial muscles and may partially or completely
eliminate the vertebral spine deformity (9). Although the
morphology of epaxial muscles stimulated by corrective LESS
has previously been described (12-14), the possible effects of
corrective electrostimulation on the specialized neuro-muscular
junction, i.e. motor end-plates (MEPs), have not yet been
determined in detail.
Therefore, the goal of the present study was to determine the
effects of short- or long-term daily LESS applied for 3 or 6
286
months on the number of motor end-plates in the longissimus
dorsi muscle (LDM) after induction of experimental scoliosis
(ES) in rabbits.
MATERIALS AND METHODS
Animal model and experimental protocol
The study was performed on thirty female rabbits, of the
New Zealand White breed, (3.5 months, weighing between 22.2 kg body weight). The animals were kept in a temperature
(18°C) and humidity (70%) controlled room. Each animal was
placed in a metal bar cage (50 cm3) with appropriate bedding
and received dry animal food and water ad libitum. The
experimental and surgical procedures described below were
approved by the Ethics Committee of Veterinary Medicine
Faculty of the University of Warmia and Mazury in Olsztyn,
Poland (decision no. 32/N/2002; and no. 2/N/2004). Electrical
stimulation of muscles was performed using a battery-operated
SCOL-2 (Elmech, Warsaw) stimulator. The stimulator’s
technical parameters were as follows: rectangular approx. 0.1
ms impulses and frequency of 20-55 Hz (15), duration of the
impulse series of 3.5-4.5 s, impulse series intervals of 4-12 s,
and the stimulation current amplitude range of 5-75 mA. The
impulse waveforms were recorded with a Tektronix
oscilloscope using a technique for imaging electrical signals.
The stimulator generated impulses had the form of subtly
differentiated rectangles, registered in a stimulator using a
substitute resistance of 5.1 kΩ, similar to human tissue
resistance (9).
Stage 1: rabbits were randomly divided into 2 groups:
control (C, n=5 animals) and experimental scoliosis (ES, n=25)
group. Rabbits from the control group were equipped with
electrodes and SCOL-2 apparatus fixed in leather harness onto
their backs, however, they were not subjected to
electrostimulation. Rabbits from ES group had the SCOL-2
apparatus electrodes attached to the thoracic region of the level
spine at Th3-Th8. To identify the particular vertebrae, animals
were shaved and X-ray images of the whole spine were taken.
After localizing the spinous processes of vertebrae of interest,
the electrodes were temporarily attached and a second X-ray
image was taken, allowing for the correction of misplaced
electrodes. Then, electrodes were fixed at particular
coordinates, marked on the animal’ skin with a permanent
marker. Either the right or left longissmus dorsi muscle (LDM),
was subjected to lateral electrical surface stimulation (LESS),
for 2 hours daily for 2 months as described previously (9). The
induction of scoliosis in each animal was assessed by x-ray
using the Cobb angle (9).
Stage 2: after 2 months of LESS, the ES group was divided
into four experimental subgroups. Two groups were subjected to
a short-term corrective electro-stimulation applied for 2 hours
daily for 3 months (I; n=5) or 6 months (II; n=5) to the
contralateral side of the spine which was not subjected to the
LESS. Another sub-group was subjected to a long-term
corrective electro-stimulation which was applied for 9 h daily for
3 months (III; n=5) or 6 months (IV; n=5), at the opposite site of
the spine.
At the end of the stimulation period, all animals belonging to
the particular experimental group were euthanized by the
overdose of thiobarbital and perfused transcardially with 4%
buffered paraformaldehyde (pH 7.4) and the longissimus dorsi
muscles were harvested from both right and left side of the
animal.
Radiologic procedure
Before the animals were randomly divided into the control
and experimental groups, all animals were anesthetized prior to
the radiological procedure by means of intramuscular injections
of metetomidine hydrochloride (0.25 mg/kg, body weight; i.m;
Cepetor; Cp-Pharma, Hendelsges, Germany). Then, an X-ray
image of the whole spine was taken in a posterior-anterior
projection in each animal and then carefully analyzed in order to
exclude any possible inborn deformations of the spine
(exclusion criterion) as well as to verify that the Cobb angle of
vertebrae of interest (i.e., Th3-Th8) was always equal (0°) (for
details, see Table 1). After this procedure, the animals were
randomly divided into the control and particular experimental
groups. At the end of each experimental procedure (i.e.,
induction of the experimental scoliosis, long- or short-term
LESS treatment), the animals of both groups were reanesthetized and an x-ray image of the whole spine was taken
again. These images were then used to evaluate and measure
(according to the Cobb method) the grade of spine deformity
induced by the initial, 2-month long LESS treatment, evoking
the experimental scoliosis. Animals that underwent the
correctional LESS treatment were then re-anesthetized again at
the end of their respective treatment protocol(s) and subjected to
a subsequent X-ray; these images allowed the quantification of
changes evoked in the Cobb angles of thoracic spine in particular
groups studied. All the measured Cobb angles in particular group
were then pooled and then presented as mean ±S.E.M. The
influence of particular correctional LESS treatment on the spine
deformity were then analyzed by using of the Student’s t test.
Table. 1. Correlation between Cobb angle, number of MEPs and fiber diameter. Abbreviations - the control (C); experimental scoliosis
(ES); short-term stimulation during 3 (I) and 6 months (II); long-term stimulation during 3 months (III) and 6 months (IV) animals
group; MEPs - motor endplates;
Animals
C
ES
I (3 months)
II (6 months)
III (3 months)
IV (6 months)
Scoliosis
average angle
±S.E.M.
0
MEPs/cm2
Fiber diameter
102 µm/10-2cm
15.36±0.49
0.66±0.01
1.1±0.04
20.31±0.56
19.6°±3.17°
Short-term correction groups
25.64±0.99
9.8°±2,91°
11.62±0.42
8.1°±3.10°
Long-term correction groups
21.0±0.92
7.9°±1.21°
8.76±0.41
8.3°±1.52°
1.14 ±0.01
1.28±0.13
1.4 ±0.15
0.75±0.08
287
Immunostaining procedure
The LDMs were postfixed in the same fixative as described
above, rinsed in 0.1 M phosphate buffer (pH 7.4; 4°C) for three
days, transferred into 18% buffered sucrose and stored in this
solution at 4°C until sectioning. 10 µm thick cryostat sections of
the LDMs were cut using a freezing microtome (Zeiss Hyrax
C60 Cryotome Germany) and processed for routine single and
double-immunohistochemical labeling.
Out of 200 consecutive 10 µm cryostat cross sections of the
same tissue block, every 20th section was mounted on a gelatincoated slide and was processed for immunofluorescence labeling
with α-bungarotoxin fluorescein isothiocynate conjugate (α-BTFITC; MOPF-1176; Merck, working dilution 1:500) and α-
bungarotoxin tetramethylrhodamine conjugate (α-BT-TMR;
MOPT-1175; Merck, working dilution 1:10,000). Additional
labeling for VAChT (vesicular transporter acetylocholine, HV007; Phoenix Pharmaceuticalis, Inc; working dilution 1:1000)
were performed. Following rinsing in PBS (3×10 min), the
preparations were incubated with FITC-conjugated donkey antigoat IgG (705-096-147, Jackson IR Lab, US) to visualize the colocalization of α-BT-TMR/VAChT. Subsequently, sections were
washed and coverslipped in a carbonate buffered glycerol, pH
8.6. In LDM sections, the number of MEPs was counted in 1 cm2
of the muscle. The preparations were viewed under an Olympus
BX51 (Olympus, Japan) fluorescence microscope equipped with
appropriate filters for FITC (B1 module, excitation filter 450480 nm) and TMR (G1, excitation filter 510-550 nm). Sample
Fig. 1. Example of
radiograph images of
the rabbits, in the
control (C; Fig. 3A);
short-term stimulation
during 3 (I group; Fig.
3B) and 6 months (II
group; Fig. 3C);
experimental scoliosis
(ES; Fig. 1D); longterm
stimulation
during 3 months (III;
Fig. 1E) and 6 months
(IV; Fig. 1F) animals
group.
The number of motor end-plates per cm2 of LDM
288
$
30
***
@
***
***
20
#/&
***
&
***
10
0
C
ES
I
II
images were acquired with AnalySIS image system software
(version 3.2; Soft Imaging System GmbH, Munster, Germany
and Cell^D Olympus; Japan).
Only the clearly visible MEPs visualized by α-bungarotoxin
staining per 150 sections MEPs were counted. These data were
pooled and expressed as means. The distance between the
selected sections of the same tissue block was set at 200 µm to
avoid the counting of the same MEP. To verify the identification
of motor endplates using bungarotoxin staining, rabbit LDM
sections were double-stained with bungarotoxin and a specific
motor endplate marker, VAChT.
Morphometric procedure
We counted the total number of α-BT-positive MEPs in the
whole cryostat section (30 cryostat sections per animal were
chosen from the total number of 600 sections) at a 200-fold
magnification. The area of 1 microscopic field was 0.12 mm2,
i.e. 0.0012 cm2. The average number of MEPs in the LDM in a
given experimental group was determined by dividing the total
number of MEPs, by the number of animals in a group by the
using formula: N=P/F, where N was the number of MEPs/cm2 in
30 cryostat sections per animal); P - the number of MEPs in
sections (n=30 per animal), F - the field of all cross-sectional
areas. The muscle fibers diameter was measured of this
experiment in the microscopic field.
Statistical analysis of immunoreactivity
The results were expressed as the mean ± S.E.M. of the total
number of MEPs per cm2 found in 150 cryostat sections per
group. The differences were statistically analyzed with ANOVA
followed by Bonferroni’s post-test for multiple comparisons.
P<0.05 was considered statistically significant.
RESULTS
Radiographic data
As revealed by analysis of the X-ray images, low-grade
scoliosis was obtained in all of the rabbits in the first stage of the
experiment (2 months of initial LESS stimulation; ES), reaching
19°±2.7°S.E.M.) of the Cobb angle (Table 1). In the second stage
of the experiment (3 and 6 months of long- and short-term
correctional LESS stimulation on the site opposite to the evoked
III
IV
Fig. 2. The number of motor endplates (MEPs) per 1 cm2 of the
longissimus dorsi muscles.
C - control, ES - experimental
scoliosis electrostimulation applied
2 h a day for 2 months; I - shortterm stimulation during 3 months
for 2 h a day; II - short-term
stimulation during 6 months for 2 h
a day; III - long-term stimulation
during 3 months for 9 h a day; IV long-term stimulation during 6
months for 9 h a day;
*** statistically significant P<0.001;
$ - P<0.001 vs. ES; # - P<0.001 vs.
I; & - P<0.001 vs. ES; @ - P<0.001
vs. II.
ES, respectively), a partial correction of ES was achieved both in
a short-term correction (STC) group and in a long-term
correction (LTC) group, Thus, the average Cobb angle in
animals treated with short-term correctional LESS decreased to
9.8°±2.91° in I group (3 months) while in II group (6 months)
this value was 8.1°±3.1° S.E.M. On the other hand, the average
Cobb angle in animals treated with long-term correctional LESS
decreased to 7.9°±1.2° in III group (3 months) and to 8.3°±1.5°
in IV group (6 months), respectively. Detailed data are presented
in Table 1.
Immunostaining and morphometric data
Fig. 2 shows the numerical density of motor endplates in the
longissimus dorsi muscle in control and experimental groups. In
control animals the average number of motor endplates was
15.36±0.49 per 1 cm2 of muscle cross-section (Fig. 3A).
LESS applied for 2 months (experimental scoliosis group),
significantly increased the number of MEPs in LDM by 32%
(20.31±0.56 per cm2 of muscle; Fig. 3D) as compared to the
intact control group (Fig. 2). The short-term corrective
electrostimulation for 3 months (I) resulted in a significant
increase by 26% (25.64±0.99, P<0.001 Fig. 1 and Fig. 2B) of the
MEPs number as compared with the ES group. On the contrary,
after 6 months of short-term stimulation (II) the number of
MEPs (11.62±0.42, Fig. 2 and Fig. 3C) decreased by 43% as
compared with the ES group, and by 24% when compared to the
intact control group.
The long-term corrective electrostimulation for 3 months
(III, Fig. 3E) did not change the numerical density of MEPs in
the LDM as compared with the ES group (Fig. 2). However,
when long-term corrective LESS was applied for 6 months the
number of MEPs in the LMD (IV; 8.76±0.41) significantly
decreased by 57% and 43% as compared to the ES group and
control animals, respectively (Fig. 2; Fig. 3F). Average muscle
fibers diameter after sort- and long-term corrective electrostimulation increased when compared to control animals (Table
1). Co-localization of bungarotoxin and VAChT was found in all
MEPs analyzed (Fig. 3G-L).
DISCUSSION
During a regular obligatory check-up, it is essential to
identify scoliosis type deformations early in children with
musculoskeletal disorders. These disorders are usually treatable
289
Fig. 3. Motor end-plates
(MEPs) of the rabbit,
immunostained for αbungarotoxin in the
control (C; Fig. 3A);
short-term stimulation
during 3 months (I
group; Fig. 3B); longterm stimulation during
3 months (II; Fig. 3C);
experimental scoliosis
(ES; Fig. 3D), shortterm stimulation during
6 months (III; Fig. 3E),
long-term stimulation
during 6 months (IV;
Fig. 3F) animals group.
The motor endplates
(MEPs) of the rabbit,
immunostained for αbungarotoxin
and
VAChT (white arrow);
C (Fig. 3G), I group
(Fig. 3H), and II group
(Fig. 3I). The motor
endplates (MEPs) of the
rabbit, immunostained
for VAChT – high
magnification (3K, L).
Scale bar =100 µm in
Fig. 3A-E; 50 µm in
Fig. 3G-H and 25 µm in
Fig. 3I-L.
by modulating the nervous system, which enables the correction
of the child’s posture and motor development (16). Frequent
electric stimulation of the proprioceptive terminals induces
plastic changes in neuronal networks and improves spinal static
and spatial body positioning (17, 18). LESS has been shown to
be effective in idiopathic scoliosis patients with a Cobb angle
between 5 to 20, in children, between 4 to 15 years old, whereas
older patients may not respond to such intervention (2). A metaanalysis of early studies indicated that LESS was ineffective in
treating scoliosis, however, the studies that were analyzed had a
marked difference in the age and severity (10, 11).
LESS treatment is typically administered for 2 hours daily for
3 months modulating the supportive steering system in rabbits
(9). Inducing tonic contraction triggers prioceptic reflex, and
modifies subspinal feedback centers. LESS can be applied to
patients between the age of 5 and 15 years before the total
ossification of the iliac cartilage plate and when the angle of
deformation is below 20° (4, 15). In neuromuscular scoliosis,
290
curve posture patterns are similar to what is seen in the most
prevalent types of adolescent idiopathic scoliosis (19).
As it may be judged from the radiological data obtained in
animals undergoing the short- versus long-term correctional
LESS, it appears that the novel (2 h/day correctional LESS)
protocol of the correctional stimulation provide better results in
studied animal model. Furthermore, when also the
histopathological findings (20) were taken into consideration,
this protocol appears to be potentially less harmful to the subject.
The rabbit is a suitable animal model for electrostimulation
studies (21). In previous studies, adolescent animals were
subjected to LESS induced right-sided deformation which
resulted in an average angle 19 on Cobb`s scale (9). Following
the corrective application of LESS, the average deformation
changed in both short- and long-term groups below 10 of Cobb`s
scale (9). The novel findings of previously study shows that the
reversibility of the changes after experimental scoliosis is
different in the mammalians (rat, rabbit, chicken, pig and goat)
(21-23). After short- and long-term corrective electrostimulation, the muscle fibers showed evidence of hypertrophy
when compared to the controls. However no significant
difference was observed between the stimulation groups. The
comparison of muscle diameters and MAPs shows that despite a
small hypertrophic increase in muscle fibre diameter, the number
of MEPs after electrical stimulation have significantly increased.
Willers et al. previously suggested that the experimental
deformation of the spine in rabbits, induced by epaxial muscles
stimulation was similar to idiopathic scoliosis observed in
children. However, it should be stressed that no attempt to
correct the evoked scoliosis was performed in this study (24).
Therefore, the results of the present study are not only in line
with the above-mentioned study (24), providing a similar picture
of spine deformity forced by initial LESS treatment, but also
extending our knowledge, that at least in the rabbit model of ES,
a correctional LESS treatment may have a beneficial influence
on the deformed spine.
Results of the present study show that experimental scoliosis
induced by LESS applied for 2 hours daily over the time of 2
months was associated with a significant increase in the number
of motor end-plates in the LDM when compared to the control
group. This is a novel finding that demonstrates the mobilization
of the respective spinal motoneurons to generate new
neuromuscular junctions. The increase in the number of motor
end-plates can be associated with the functional compensation
caused by an increased control of muscles stimulated by LESS.
The pre and postsynaptic abnormality are suggested of an
impaired of the neuromuscular junction and imply the motor
neurons plays an important role in ensuring proper functions of
the neuromuscular synapses (25). Moreover, it has been reported
that increased neuromuscular activity is an important for motor
end-plates.
Neuromuscular junctions have the ability to adjust to various
levels of activity through morphological and physiological
adaptation which include the sprouting of nerve terminals or the
reduction of the endplate surface area (26). However, the
increased neuromuscular activity associated with repeated
periods of endurance (27) and resistance exercise (28), results in
the significant expansion of pre- and post-synaptic
neuromuscular junctions.
Thus, the changes observed in our study, concerning both
the diameter of muscle fibres challenged by LESS as well as in
the number of MEPs observed on the stimulated muscle,
corroborate the view that the skeletal muscle tissue posses a
considerable potential for adaptation to external factors
influence its homeostasis. This observation appears to be in the
similar with other studies, showing that even foreign body implants mimicking an ectopic bone tissue, were able to induce
profound changes in genitive machinery of afflicted muscle in
the rat (29, 30).
Interestingly, the corrective electrostimulation of the
contralateral muscles in rabbits with experimental scoliosis
increased further the number of MEPs provided that it did not
lasted too long on both the daily and monthly basis. This
phenomenon seems to be important for the positive effects of
the corrective electrostimulation since the rise in the MEPs
number should provide a better control of paravertebral muscles
by the central nervous system. However, this positive result of
corrective LESS has clear limitations related to its duration,
since the prolongation of LESS to 6 months produced
completely adverse effects on the number of MEPs as compared
with 3 months of LESS application. One can hypothesized that
the multiplication of repeated electrostimulation leads to the
exhaustion of the regenerative properties of spinal motoneurons
what results in the profound decrease of MEPs in the stimulated
paraspinal muscles. Significant decrease in the number of
neuromuscular junctions at the side corrected for 6 months by
LESS in II and IV groups suggests an exhaustion of
compensatory mechanisms, indicating that the overburdening
phenomenon may have occurred. However to verify this,
further studies of the structure and function of motor neuron of
spinal cord would be necessary. Future research should examine
the distribution of motor neurons supplying the LDM within the
spinal cord of the rabbit as previously examined in the pig (31).
Previous experiments also showed that the LESS method
applied for 9 hours causes deformities of the muscular and
connective tissues stabilizing the spine which leads to
secondary spine destabilization (12, 14, 32). Application of
short-term LESS did not lead to such changes, but instead lead
to an observed increase in muscular fibers, multiplication and
increase in their activity (32, 33). After the analysis of the
results of the experiments with rabbits, it was found that longterm LESS may cause neurogenic and miogenic lesions as well
as an overload of the muscle which stabilize the spine (34).
Such over-stimulation induced changes may account for
failures in LESS therapy (16).
The increase of MEPs in the paravertebral muscles
following a shorter exposure time to LESS suggests that shorter
periods of corrective electrostimulation may be employed as an
additional and noninvasive treatment of scoliosis following
further research.
Conflict of interests: None declared.
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R e c e i v e d : February 14, 2012
A c c e p t e d : June 18, 2012
Author’s address: Dr. Joanna Wojtkiewicz, Department of
Neurology and Neurosurgery, Division of Neurosurgery, Faculty
of Medical Sciences, University of Warmia and Mazury, 30,
Warszawska Street, 10-082 Olsztyn, Poland;
E-mail: [email protected]