ELECTRICAL STIMULATION IMPAIRS EARLY FUNCTIONAL RECOVERY
AND ACCENTUATES SKELETAL MUSCLE ATROPHY AFTER SCIATIC
NERVE CRUSH INJURY IN RATS
DAVILENE GIGO-BENATO, MD,1 THIAGO LUIZ RUSSO, PhD,2 STEFANO GEUNA, PhD,3 NATALIA REZENDE SANTA ROSA
DOMINGUES,1 TANIA FÁTIMA SALVINI, PhD,2 and NIVALDO ANTONIO PARIZOTTO, PhD1
1
Unit of Thermophototherapy, Department of Physical Therapy, Federal University of São Carlos, Rodovia Washington Luis, Km 235,
CP 676, CEP 13.565-905, São Carlos, SP, Brazil
2
Unit of Skeletal Muscle Plasticity, Department of Physical Therapy, Federal University of São Carlos, São Carlos, SP, Brazil
3
Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Regione Gonzole, Orbassano, Italy
Accepted 17 August 2009
ABSTRACT: Neuromuscular recovery after peripheral nerve
lesion depends on the regeneration of severed axons that re-
establish their functional connection with the denervated mus-
cle. The aim of this study was to determine the effects of elec-
trical stimulation (ES) on the neuromuscular recovery after
nerve crush injury in rats. Electrical stimulation was carried out
on the tibialis anterior (TA) muscle after sciatic nerve crush
injury in a rat model. Six ES sessions were administered every
other day starting from day 3 postinjury until the end of the
experiment (day 14). The sciatic functional index was calcu-
lated. Muscle excitability, neural cell adhesion molecule (N-
CAM) expression, and muscle fiber cross-sectional area (CSA)
were accessed from TA muscle. Regenerated sciatic nerves
were analyzed by light and confocal microscopy. Both treated
(crushþES) and untreated (crush) groups had their muscle
weight and CSA decreased compared with the normal group (P
< 0.05). Electrical stimulation accentuated muscle fiber atrophy
more in the crushþES than in the crush group (P < 0.05).
N-CAM expression increased in both crush and crushþES
groups compared with the normal group (P < 0.05). Regener-
ated nerves revealed no difference between the crush and
crushþES groups. Nevertheless, functional recovery at day 14
post-injury was significantly lower in crushþES group compared
with the crush group. In addition, the crushþES group had
chronaxie values significantly higher on days 7 and 13 com-
pared with the crush group, which indicates a decrease in mus-
cle excitability in the crushþES animals. The results of this
study do not support a benefit of the tested protocol of ES dur-
ing the period of motor nerve recovery following injury.
Muscle Nerve 41: 685–693, 2010
Nerve lesions represent a high cost for society.1
Nerve lesions result from traumas that range
from simple nerve fiber compression to complete
transection.2 When nerve fiber continuity is inter-
rupted, the distal stump undergoes a particular
series of tissue changes known as Wallerian degen-
eration.3 The proximal axon stump can then
regenerate along the nerve tract distal to the lesion
and reach the peripheral target (muscle fibers and
sensory receptors), leading to recovery of func-
tion.3 Furthermore, many strategies are performed
by skeletal muscle after nerve injury to promote
muscle fiber reinnervation, such as the increase of
neural cell adhesion molecule (N-CAM) expression
Abbreviations: CSA, cross-sectional area; ES, electrical stimulation;
N-CAM, neural cell adhesion molecule; SFI, sciatic functional index; TA,
tibialis anterior
Key words: electrical stimulation nerve injury; neuromuscular impairment;
rehabilitation; skeletal muscle
Correspondence to: N.A. Parizotto; e-mail: parizoto@ufscar.br
V
C 2010 Wiley Periodicals, Inc.
Published online 15 May 2010 in Wiley InterScience (www.interscience.
wiley.com). DOI 10.1002/mus.21549
Electrical Stimulation and Nerve Injury
around and inside the muscle fibers.4 N-CAM is a
robust candidate for use in the investigation of the
interaction between muscle and nerve.
The degree of tissue recovery can be highly
variable,5,6 however, and it is thus very important
to seek effective posttraumatic physiotherapeutic
protocols to improve the final degree of functional
recovery in patients with a peripheral nerve
lesion.6,7 One of the therapeutic approaches pro-
posed to improve posttraumatic neuromuscular
recovery is electrical stimulation (ES), applied
either to the severed nerve8,9 or to the denervated
muscles.1,10–12 With regard the latter approach,
experimental studies carried out so far have pro-
duced contradictory results. Although some investi-
gators have shown that muscle ES improves nerve
regeneration,11 others found no difference in the
extent of regeneration between treated and
untreated groups.13
To further understand this issue, we aimed to
investigate early functional and morphological
changes induced by muscle ES, guided by muscle
electrical excitability evaluation and applied by
surface electrodes as normally used in the rehabil-
itation of denervated muscles in humans, after
application of a standardized crush lesion in the
rat sciatic nerve model, a well-described and con-
trolled model of axonotmesis.14 This type of
lesion is characterized by the beginning of func-
tional recovery as early as the second posttrau-
matic week, as detected by walking track analysis,
and is thus particularly suitable for investigating
early nerve recovery. The main purpose of the
present study was to determine whether a clinical
protocol of ES could stimulate neuromuscular
recovery.
METHODS
Animal Care and Experimental Groups. Twenty-
seven, 3-month-old, male Wistar rats (298 6 1 g)
were used in this study. Good laboratory animal
practices were observed in accordance with the
international standards for animal experimentation
and following approval by the local institution’s
MUSCLE & NERVE May 2010 685
animal care and ethics committee. Animals were
divided into three groups (n ¼ 9): (1) crush, ani-
mals had their right sciatic nerve crushed; (2)
crushþES, the right sciatic nerve was crushed, and
the tibialis anterior (TA) muscles were submitted
to muscle excitability evaluation and electrical stim-
ulation every other day; and (3) normal (N), con-
trol animals, with no procedures performed.
Nerve Injury Procedure. For inducing the crush
lesion, rats were anesthetized with an intraperito-
neal injection of a premixed solution containing
ketamine (95 mg/kg) plus xylazine (12 mg/kg).
The skin was shaved and cleaned with 10% povi-
done iodine. A 2-cm-long incision was made on
the skin through a gluteal approach and the left
sciatic nerve was exposed. A non-serrated clamp,
exerting a force of 54 N,15 was applied for a period
of 30 seconds to create a 3-mm-long crush injury,
10 mm above the bifurcation, to obtain an axo-
notmesis lesion that has been shown to be repro-
ducible.14 The starting diameter of the sciatic
nerve was about 1 mm. During the crush, the
nerve flattened to a new diameter of 2 mm, giving
a final pressure of 9 MPa. The nerves were kept
moist with 37
C sterile saline solution throughout
the surgical intervention. The fascia and skin were
then sutured in a distal-to-proximal manner using
silk thread. After surgery, animals were housed in
single cages and fed rat chow and water ad libi-
tum. For the first 4 days, acetaminophen was
added to the animals’ drinking water for pain
reduction.
Electrical Evaluation and Electrical Stimulation Proce-
dures. Rats from the crushþES group received ES
treatment as previously described.12,16 The animals
were anesthetized during all electrical procedures.
ES equipment that permits changes in the electri-
cal parameters was used to assess muscle excitabil-
ity and also for the ES treatment. Briefly, before
fixing the electrodes, the skin was shaved and
cleaned and a gel conductor layer applied between
the electrodes and skin. Two electrodes were used.
The anode electrode (rubber circular self-adhesive
electrode, 5 cm in diameter) was positioned on
the animal’s back; it had a large area, providing a
decrease in concentration of the electrical charge
on the skin, and there was no hyperemia observed
after the electrical procedures. The cathode elec-
trode (a metallic electrode, 3 mm in diameter) was
used to stimulate the TA muscle; it was small
enough to stimulate only the TA muscle. During
the ES procedure, this electrode was maintained in
close proximity to the skin over the TA muscle.
Furthermore, surface electrodes are more com-
monly used for therapeutic applications, and their
686 Electrical Stimulation and Nerve Injury
position does not require procedures that are inva-
sive to ES.17
Before each evaluation of electrical parameters,
we identified the site over the TA where the lowest
stimulus amplitude fully activated the muscle. The
electrical parameters were evaluated before each
ES treatment to provide rheobase, chronaxie, and
muscle accommodation values. Afterwards, the
chronaxie values were used to determine the ES
parameters applied to the TA muscle. These elec-
trical indexes were previously reported16,18 to be a
guide for selection of the parameters for ES.
Briefly, rheobase is the minimal electrical stimulus
intensity necessary to produce a muscle contrac-
tion using a rectangular phasic current with
greater pulse duration. Chronaxie is the minimal
pulse duration necessary to induce a muscle con-
traction (rectangular phasic current, amplitude of
twice the rheobase value). Finally, accommodation
is the muscle’s capacity to not respond to slowly
incrementing electrical pulses (exponential phasic
current). Crush group excitability was also investi-
gated, and it was submitted to four evaluations: im-
mediately before and after nerve injury, and then
7 and 13 days after nerve injury. These values
allowed us to observe the excitability evolution dur-
ing neuromuscular recovery in the crush group.
Values identified during the electrical evalua-
tion were used to determine the ES parameters for
each session, as previously described.18 For chron-
axie values >1 ms, an exponential phasic current
was used (frequency: 20 HZ; pulse duration: twice
the chronaxie value; time on: 3 s/time off: 6 s).
The amplitude of pulse necessary to induce a visi-
ble contraction was selected. The selected stimula-
tion frequency permitted strong muscle contrac-
tion using low current amplitudes, as previously
described.20
After muscle excitability evaluation, the ampli-
tude necessary to induce maximal contraction of
the TA muscles was identified. This maximal con-
traction was considered when full flexion of the
right ankle was seen. Each ES session produced 20
maximal contractions of the TA muscle, applied
every 48 h for 14 days, beginning 72 h after dener-
vation. Thus, animals from the crushþES group
received six sessions of muscle excitability evalua-
tion and ES.
In our study, a small number of muscle con-
tractions were used to mimic what is usually under-
taken during a single session of rehabilitation for
the recovery of denervated human muscles. Nor-
mally, in a single session of treatment, the electri-
cal treatment of denervated muscles is associated
with other interventions, such as physical exercise,
muscle stretching, and passive movements. More-
over, we decided upon 20 muscle contractions,
MUSCLE & NERVE May 2010
because the long duration of stimulation used
(time on ¼ 3 s) could provoke muscle fatigue, as
all contractions were made in a short time period
during a single treatment session.
Motor Function Evaluation. The assessment of
nerve function recovery was carried out by calculat-
ing the sciatic functional index (SFI), as described
by Bain et al.21 Animals were tested in a confined
walkway that was 42 cm long and 8.2 cm wide, with
a dark shelter at the end. A white paper was placed
on the floor of the rat walking corridor. The hind
paws of the rats were pressed down onto a finger
paint–soaked sponge, and the animals were then
allowed to walk down the corridor leaving hind
footprints on the paper. Three measurements were
taken from the footprints: (1) the print length
(PL), which is the distance from the heel to the
third toe; (2) the toe spread (TS), which is the dis-
tance from the first to the fifth toe; and (3) the
intermediary toe spread (ITS), which is distance
from the second to the fourth toe. All three meas-
urements were taken from the experimental (E)
and normal (N) sides. The SFI was calculated
according to the following equation:
   
EPL  NPL ETS  NTS
SFI ¼ 38:3 þ 109:5
 NPL  NTS
EITS  NITS
þ 13:3  8:8
NITS
Two weeks after surgery, all animals were eutha-
nized with anesthesia overdose and the sciatic
nerves and the TA muscles were carefully dissected
from the operated right side to avoid mechanical
injuries. To have a first indication on trophic varia-
tions, wet muscles were immediately weighed with
a precision balance (Model 100a; Denver Instru-
ments, Denver, Colorado).
Muscle Evaluation. TA muscles were then frozen
in isopentane, previously frozen in liquid nitrogen.
Muscle samples were stored in a freezer at 80
C.
Histological serial cross-sections (10 lm), cut trans-
versely to the muscle main axis, were obtained
with a cryostat (HM 505E; Microm, Walldorf, Ger-
many) at a level corresponding to the middle belly
of the muscle. Sections were stained with toluidine
blue for morphological evaluation. For muscle
fiber morphometry, the cross-sectional area of 100
randomly selected fibers was measured in the mid-
dle belly of each TA muscle, using a light micro-
scope (Axioplan 2; Carl Zeiss, Jena, Germany)
equipped with a digital camera (DSC S75; Sony,
Tokyo, Japan) and AxioVision software (Carl Zeiss,
Jena, Germany). A blinded procedure was used for
the measurements.
Electrical Stimulation and Nerve Injury
Rabbit anti–N-CAM affinity-purified (1:100 dilu-
tion; Catalog No. AB5032; Chemicon Interna-
tional, Temecula, California) primary antibody and
the secondary antibody rhodamine red goat anti-
rabbit IgG (1:150 dilution; Catalog No. Rb394;
Molecular Probes, Eugene, Oregon) secondary
antibody were used for immunostaining. The cross-
sections of muscles used for immunostaining
against the N-CAM were fixed with 4% paraformal-
dehyde in 0.2 M phosphate buffer (PB) for 10 min
at room temperature, blocked with 0.1 M glycine
in PBS for 5 min, and permeabilized in 0.2%
Triton X-100–PBS for 10 min. The slides were
incubated with a solution containing the primary
antibody, 3% normal goat serum, and 0.3% Triton
X-100–0.1 M PB overnight in a moisture chamber
(4
C). After washing the slides with 0.1 M PB
(3 times for 10 min each), we added a solution
containing the secondary antibody and 0.3%
Triton X-100–0.1 M PB for 2 h in a dark room.
The slides were washed in 0.1 M PB (three times
for 10 min each). The slides were mounted with
mounting medium for fluorescence with 4,6-diami-
dino-2-phenylindole (Catalog No. H-1200; Vecta-
Shield; Vector Laboratories) applied to the cover-
slips. Observations were made by obtaining
photomicrographs of the stained sections using a
fluorescence microscope (Axiolab; Carl Zeiss, Jena,
Germany) equipped with a rhodamine filter. Both
20 and 40 magnifications were used. For quan-
titative measurements of N-CAM immunoreactivity,
images of five different regions from the middle
belly of the TA muscles were captured (Axiocam;
Carl Zeiss) at a final magnification of 20, with
the microscopic setting kept the same for all slides.
Quantification was performed by ImageJ software
using the tool color histogram (version 1.41o;
Wayne Rasband, National Institutes of Health,
Bethesda, Maryland; http://rsb.info.nih.gov/ij).
Nerve Evaluation. The sciatic nerves were fixed in
10% formalin for 3 h and then washed in phos-
phate-buffed saline (PBS) until embedding. The
specimens were dehydrated and embedded in
paraffin and cut at 7 lm perpendicular to the main
nerve axis. For light-microscopic analysis, sections
were stained with hematoxylin and eosin and Papa-
nicolau stain and observed with a Leica DM400
microscope equipped with a Leica DFC320 digital
camera. For immunohistochemistry and confocal
laser microscopy, sections were incubated overnight
with the anti-neurofilament 200 kDa (mouse mono-
clonal, which recognizes the pig 200-kDa subunit of
neurofilaments, at 1:200 dilution; Sigma, St. Louis,
Missouri). After washing in PBS, immunolabeling
was carried out by incubating the sections for 1 h
with goat anti-mouse Alexa-Fluor 488–conjugated
MUSCLE & NERVE May 2010 687

FIGURE 1. Functional deficit in the crush and crushþES
groups, assessed by sciatic functional index (SFI) calculation.
In the pre-lesion state, function was considered normal in both
groups. In the first week after injury the values were lower; in
the second week, the SFI for crushþES group remained close
to 80, whereas SFI in the untreated group was around 60.
expected, the beginning of functional recovery, that
is, a mean SFI (67.4 6 16.5) significantly (P <
0.05) different than that at day 7, crushþES animals
still showed a mean SFI of 88.9 6 13.1, which was
not significantly (P < 0.05) different from day 7,
thus pointing to the persistence of almost complete
impairment of sciatic nerve function.
Rheobase, chronaxie, and accommodation val-
ues obtained from the right TA muscles immedi-
ately before nerve crush (IBC) in the crush and
crushþES groups were considered as normal mus-
cle excitability values (Fig. 2A–C). Rheobase

IgG (dilution 1:200; Molecular Probes, Eugene,

Oregon). The sections were finally mounted with a
Dako fluorescent mounting medium and analyzed
by a confocal laser microscopy system (LSM 510;
Zeiss, Jena, Germany), which incorporates two
lasers (argon and helium–neon), and is equipped
with an inverted Axiovert 100M microscope.
Confocal fluorescence images were taken using a
20 Plan-Neofluar objective with a numerical aper-
ture (NA) of 0.50 and a 40 Plan-Neofluar objec-
tive with a NA of 0.75. An electronic zoom with a
magnification ranging from 1 to 8 was employed to
obtain the magnifications indicated in the figures.
To visualize Alexa-Fluor 488 fluorescence, we used
excitation from a 488-nm argon laser line and emis-
sion passing through a band-pass (BP) 505–530 fil-
ter, which passes wavelengths at 505–530 nm to the
detector.

Statistics. All numerical data were subjected to

statistical analysis by one-way analysis of variance
(ANOVA), followed by post hoc multiple pairwise
comparisons carried out using Tukey’s test. Statisti-
cal significance was established at P < 0.05.

RESULTS
Figure 1 shows the results of the functional assess-
ment of posttraumatic sciatic nerve recovery.
Statistical analysis showed that, as expected, the dif-
ferences in SFI between the pre-lesion (normal
walking) and both post-lesion assessments were stat-
istically significant (P < 0.05) for the two experi-
mental groups (crush and crushþES). Statistical
comparison between the two experimental groups
showed the presence of no significant (P > 0.05) dif-
ference in the pre-lesion baseline evaluation and in
the assessment made at day 7 post-crush, when SFI
values approximated 90, indicating complete ab-
sence of sciatic nerve function. On the contrary,
there was a significant (P < 0.05) difference
between crushþES and crush animals at day 14 post-
crush: Whereas the crush group showed, as
FIGURE 2. Tibialis anterior (TA) muscle excitability representa-
tion throughout the nerve injury period. Data are the mean 6
standard deviation. IBC, immediately before crush; IAC, imme-
diately after crush. IBC values were considered normal control
values. (A) Rheobase and (C) accommodation: *P < 0.05 when
crush and crushþES are compared with their own IBC values.
(B) Chronaxie: *P < 0.05 when both crush and crushþES
groups are compared with their own IBC values; †P < 0.05
when crush was compared with crushþES at 7 and 13 days.
Note that both rheobase and accommodation decreased after
nerve injury. Chronaxie increased significantly in denervated
muscles submitted or not to ES, and crushþES presented
higher chronaxie values compared with crush at days 7 and 13.

688 Electrical Stimulation and Nerve Injury MUSCLE & NERVE May 2010
decreased 13 days after nerve injury in the
crushþES animals compared with the group’s IBC
values (P < 0.05; Fig. 2A). This decrement started
on day 3 after nerve injury and remained
unchanged from 5 to 13 days (P > 0.05). The
rheobase values from the crush group did not dif-
fer from those found in crushþES at days 7 and 13
(P > 0.05; Fig. 2A). Similar to rheobase, accommo-
dation values decreased after nerve injury in both
crushed groups (submitted or not to ES) com-
pared with IBC accommodation levels (P < 0.05),
and no difference was observed between them at
days 7 or 13 (P > 0.05; Fig. 2C). On the other
hand, chronaxie increased exponentially along the
first week after nerve injury in both crush and
crushþES groups (Fig. 2B) compared with IBC val-
ues (P > 0.05). Despite this, crushþES showed
chronaxies significantly higher than those of the
crush group on days 7 and 13 (P < 0.001; Fig. 2B).
Both crush and crushþES groups decreased
their muscle weights compared with the N group (P
< 0.05), with no difference between them (P >
0.05). Figure 3 shows the microscopic appearance
of TA muscles (on the left side) and their respective
muscle fiber CSA distribution (on the right side) in
N (Fig. 3A and B), crush (Fig. 3C and D), and
crushþES (Fig. 3E and F) animals, respectively.
Muscle fiber atrophy was evident in both crushed
groups (Fig. 3C and E) compared with the N group
(Fig. 3A). Muscle fiber CSA measurement con-
firmed the atrophy (Fig. 3D and F). Muscles from
crushþES animals had >80% of the total number of
muscle fibers, with the CSA ranging from 501 to
1000 lm2 (Fig. 3F), whereas the muscles from the
crush animals had approximately 40% of the muscle
fiber CSA ranging from 501 to 1000 lm2 and the
other 45% from 1001 to 1500 lm2 (Fig. 3D). Statis-
tics for muscle fiber CSA mean values confirmed the
distribution. CrushþES and crush groups both
showed a significant reduction, of about 55% and
38% of the muscle fiber CSA, respectively, com-
pared with N (P < 0.05). The muscle fiber CSA of
crushþES animals was significantly smaller than
that of crush animals (P < 0.05).
Expression of N-CAM was restricted to the
neuromuscular junction in the N group (Fig. 4,
top left). Denervated muscles from the crush (Fig.
4, top right) and crushþES (Fig. 4, lower left)
groups increased expression of N-CAM, which was
observed around atrophied muscle fibers, with no
difference in expression between the groups
(Fig. 4). Quantitative analysis of N-CAM immuno-
reactivity confirmed this increase in the crush
and crushþES groups compared with N (P < 0.05;
Fig. 4, lower right). No difference was observed
between injured groups (P ¼ 0.89).
Electrical Stimulation and Nerve Injury
Figure 5 shows light (Fig. 5A, C, and E) and
confocal (Fig. 5B, D, and F) imaging of sciatic
nerve bundles from the N (Fig. 5A and B), crush
(Fig. 5C and D), and crushþES (Fig. 5E and F)
groups. As expected, the differences between nor-
mal and regenerated nerves were evident. On the
other hand, a comparison between crush and
crushþES nerves indicated no major difference
with regard to histological organization (Fig. 5C
and E) of the nerve or morphology of regenerat-
ing axons labeled by anti-neurofilament 200 kDa
(Fig. 5D and F).
DISCUSSION
The objective of this study was to investigate the
early effects of ES by surface electrodes on neuro-
muscular recovery after a standardized crush
injury. Results show that morphology of regener-
ated nerve fibers and N-CAM expression in dener-
vated muscles were similar in the crushþES and
crush groups. Nevertheless, ES impaired the neu-
romuscular functional recovery and accentuated
muscle fiber atrophy in the crushed sciatic nerve
model. In fact, although early signs of functional
recovery measured by the sciatic functional index
could be observed in the crush group, no recovery
was noticed in treated animals (crushþES). Fur-
thermore, chronaxie values remained higher in
the crushþES than in the crush group, indicating
that ES could interfere with muscular excitability
recovery after denervation.
These results support the observations of other
experimental studies,19 which showed that the ES
delays nerve regeneration after crush lesions in
mice. The investigators19 reported that transcuta-
neous electrical nerve stimulation (TENS) led to
nerves having morphological signs of impaired
regeneration, such as more axons with dark axo-
plasm, signs of edema, and a less organized
cytoarchitecture, after crush injury in mice. More-
over, fewer and thinner myelinated fibers and an
increased number of Schwann cell nuclei were also
observed in the groups submitted to TENS. It is
relevant to note that the TENS was applied by
using surface electrodes similar to those in our
protocol of stimulation.
It seems that different aspects of ES, such as
type of stimulation (using implanted or surface
electrodes), time of stimulation, frequency, and
direct stimulation of the nerve or muscle, are rele-
vant in the impairment or improvement of nerve
recovery. Although the present study showed that
ES applied by surface electrodes may weaken func-
tional recovery and accentuate muscle atrophy af-
ter nerve crush injury, other studies10,22,23 reported
that ES improved nerve regeneration. Mendonça
MUSCLE & NERVE May 2010 689
FIGURE 3. Histological cross-sections of tibialis anterior muscle fibers stained by toluidine blue and their respective cross-sectional
area (CSA) distribution. (A, B) N group, (C, D) crush group, and (E, F) crushþES group. Scale bars ¼ 40 lm. Both denervated
muscles (crush and crushþES) had decreased muscle fiber CSA. However, the muscle fiber CSA distribution levels of denervated
muscles submitted to ES were significantly smaller than those not stimulated and denervated (crush).

et al.24 showed that low-intensity ES, applied
directly to the nerve by implanted electrodes,
enhanced both morphologic and functional regen-
eration of crushed sciatic nerves in rats. They
attributed the mechanism of ES action to a proba-
ble delay in axonal degeneration, stimulating nerve
sprouting, and accelerating myelin sheath regener-
ation.24 ES by implanted electrodes also showed
beneficial effects that accelerated facial nerve
recovery in the rat model.25 Lu et al.26 observed
690 Electrical Stimulation and Nerve Injury
that low frequencies of stimulation (2 HZ) pro-
mote regeneration, with transected nerves reveal-
ing more myelinated fibers, higher axon density,
and higher ratio of blood vessel to total nerve area
compared with the non-stimulated and injured
nerves.
It should also be pointed out that Kerns and
Lucchinetti22 found significant improvement in
the twitch tension of crushed nerves only when ES was
applied during the middle period (days 12–21 after
MUSCLE & NERVE May 2010
FIGURE 4. Neural cell adhesion molecule (N-CAM) immunofluorescence from TA muscles. Representative micrographs showing the
expression of N-CAM restricted to the neuromuscular junction (white arrows) in the N group, and around atrophied muscle fibers in
the crush and crushþES groups (scale bar ¼ 100 lm). Quantitative estimates of levels of N-CAM immunoreactivity showed an
increase of N-CAM in both crush and crushþES groups when compared with N. *P < 0.05 compared with N. [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]

nerve crush), and no differences were noted at either
the early or later stages of recovery. These observations
suggest that direct muscle ES may exert a stimulatory
effect on nerve regeneration only in a specific window
of time during the regeneration process—not earlier
or later. Our data appear to support the idea that if
muscle ES is administered very early after the nerve
lesion, when axons are still being renewed along the
distal nerve stump not reaching the muscle fibers, the
ES may even exert an inhibitory effect on the func-
tional neuromuscular recovery, which can also be con-
firmed by muscle excitability changes due to denerva-
tion. Muscles submitted to ES required a greater
width of electrical pulses to provoke a contraction
compared with muscles in the crush group, indicating
a decreased velocity of electrical conduction in the
crushþES and crush group animals.
Although modifications to muscle excitability
were significant, N-CAM expression was not
impaired by ES. Recently, Kostrominova and Cols27
showed that denervated muscles submitted to ES
(applied chronically and using implanted electro-
des) did not express N-CAM in those muscle fibers
in which the trophism was maintained by ES.
Electrical Stimulation and Nerve Injury
N-CAM expression was restricted to the neuromus-
cular junction, satellite cells, and some atrophied
muscle fibers. In the future, other factors related
to muscle reinnervation, such as expression of
acetylcholine receptors and agrin, should be inves-
tigated to verify whether the present ES protocol
can regulate these others factors of reinnervation
and whether short- and long-term ES protocols
present different responses with regard to neuro-
muscular recovery.
Experimental aspects, such as difficulty in repro-
ducing a standardized nerve lesion,14 in the differ-
ent studies dealing with nerve regeneration should
be compared carefully. To address this issue, in the
present study we employed a device for inducing a
reproducible sciatic nerve crush injury, which has
been recently realized15 and standardized.14 The
results of our study may thus establish a compara-
tive baseline for future studies for ours and other
laboratories aiming to investigate the effects of ES
(as well as of any other type of treatment) on post-
traumatic nerve regeneration.
Another major aspect of the present study was
to verify whether a clinical-like protocol of ES,
MUSCLE & NERVE May 2010 691
FIGURE 5. Light (hematoxylin and eosin) and confocal (anti-neurofilament 200-kDa immunostaining) imaging of sciatic nerve from N
(A, B), crush (C, D), and crushþES (E, F) group animals. Scale bars ¼ 20 lm. The crushþES and the crush group nerves showed
no difference between them, but they did show a structural and morphological difference when compared with the normal group. Black
and white arrows indicate normal axonal structure.

similar to what is normally used in rehabilitation
of denervated muscles in humans, could exert any
effects on neuromuscular recovery. Other studies
have shown that ES is a potent stimulus able to
detain degeneration in denervated muscle28 as well
as regulate molecular changes in skeletal muscle
due to peripheral nerve injuries.12,16,27 Further-
more, the amount of stimulation28 and the interval
between each stimulation29 were found to be the
main factors in the prevention muscle atrophy.
However, those studies27–29 were performed in dif-
ferent models of nerve injury, such as total nerve
section, using implanted electrodes to stimulate
692 Electrical Stimulation and Nerve Injury
the skeletal muscle,30 and applying ES in a chronic
way (entire day and during many weeks, not in sin-
gle sessions). Besides, in general, they did not
investigate the ES effects on the peripheral nerv-
ous system. Evaluation of ES protocols similar to
those applied in rehabilitation can bring relevant
information to the development of effective and
safe therapies for treating denervated or recover-
ing muscles, with no harmful effects to potential
nerve regeneration.
Furthermore, there are obvious ethical reasons
contraindicating both nerve and muscle biopsies
in individuals with peripheral nerve injury, which
MUSCLE & NERVE May 2010
shows the importance of controlled animal models,
such as the one used herein, to study neuromuscu-
lar recovery after axonotmesis. On the other hand,
studies involving both imaging and functional anal-
yses in humans are fundamental to correlation of
data obtained in animals to humans.
In conclusion, our data suggest that the effec-
tiveness of electrical stimulation on posttraumatic
nerve recovery remains open to question and, if
not properly used in terms of stimulation protocol
and time of application, this type of physical ther-
apy may be not only ineffective but may even in-
hibit the repair process that brings a relevant con-
tribution to the skeletal muscle rehabilitation area.
Therefore, future studies need to define the clini-
cal conditions and treatment protocols that
can improve clinical outcome among patients with
peripheral nerve trauma.
This work was supported by grants from the Italian Ministry of
Education, University and Research (MIUR). The authors thank
Dialogue International, Torino, Italy, for English language revi-
sion. D.G.-B. and T.L.R. are doctoral and postdoctoral grant hold-
ers from Fundação de Âmparo à Pesquisa do Estado de São Paulo
(FAPESP, Proc. 06/52931-4 and 08/05237-0, respectively).
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