Electrical stimulation of ligated sciatic nerve in mice

Eur J Transl Myol 27 (3): 173-182

Effect of electrical stimulation on motor nerve regeneration in sciatic

(1) Biomedical Engineering Faculty, Science and Research Branch, Islamic Azad University,
Tehran, Iran; (2) Department of Pharmacology, School of Medicine, Tehran University of
Medical sciences, Tehran, Iran
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits
any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract
The purpose of this study was to investigate the effect of electrical stimulation on sciatic nerve
regeneration and functional recovery of target muscles. Mice were randomly divided into 3
groups: ligated without electrical stimulation, ligated with electrical stimulation and control
(non-ligated). The unilateral peripheral mononeuropathy was produced on the right hind limb.
Sciatic nerve was then electrically stimulated daily for a period of 2 weeks (duration: 0.2 msec,
frequency: 100Hz, amplitude: 15mA). Evoked surface EMG was recorded from biceps femoris
(BF) and gluteus maximus (GM) muscles on the 3rd, 7th, 10th and 14th day after sciatic nerve
ligation. Muscle force and sensitivity was determined by processing of the recorded EMG
signals in time and frequency domains respectively. The results showed electrical stimulation
(ES) produced a significant increase in the EMG response of BF, and muscle force
significantly increased on the 14th day (p<0.001), however no significant difference was found
in GM muscle force between experimental groups. This may be due to possible innervation by
inferior gluteal nerve. Frequency analysis of BF signals indicates that hyperalgesia remained
after 14 days in both ligated groups. On the 14th day no difference in GM muscle sensitivity
was found between groups. In conclusion, the results of this study have shown that the
electrical stimulation of sciatic nerve accelerates nerve repair and indirectly improves BF
muscle force to a comparable level with control without effect on muscle sensitivity. However,
ES had no effect on GM muscle force and sensitivity.
Key Words: EMG, electrical stimulation, muscle force, muscle sensitivity, ligated mice
Eur J Transl Myol 27 (3): 173-182

Partial or complete loss of sensory and motor function recovery following nerve injury, have focused on
may be induced by injury to peripheral nervous system. electrical stimulation of muscles,12,13,29 or
10
Peripheral nerves are able to regenerate following lesion neuromuscular junction. Effect of nerve electrical
or disease. However, regeneration is not complete and stimulation on related muscle electrophysiology has
the function rarely returns to pre-injury levels.1,2,3 however received less attention. Surface
Depending on the age and the severity of the lesion, the electromyography (SEMG) is a non-invasive method
nerve repair is limited and different.4,5 Electrical which is an indirect indicator of the central drive. On
stimulation (ES) is a popular treatment, useful to the other hand, this method is directly associated with
recover from muscle atrophy and also used to evoke activity of the motor units which are affected by
functional contractions. Different types of ES such as physiological parameters such as motor unit discharge
weak direct current,6 high-voltage electrical currents,7 rates, and muscle fiber membrane characteristics.15,16
alternating currents,8 transcutaneous nerve stimulation,9 So, the purpose of this study was to investigate the
neuromuscular electrical stimulation,10 and effect of electrical stimulation on sciatic nerve
11
electromagnetic fields are also used to promote nerve regeneration and functional recovery of target muscles.
regeneration and to improve neuromuscular functions, Materials and Methods
but their precise effects on nerve regrowth are debated. Animals
Recent studies in the field of peripheral nerve repair are
focused on histological methods. 2,7,9 Furthermore, a Male NMRI mice (20-25g) were housed in groups of 10
number of investigations regarding neuromuscular in conditions of constant temperature (20±2℃) with

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 Electrical stimulation of ligated sciatic nerve in mice
Eur J Transl Myol 27 (3): 173-182

Fig 1. Diagram of the experimental set-up.

12L:12D photoperiod, and were allowed free access to peripheral mononeuropathy to study regeneration of
food and water except during the experimental sessions. peripheral nerve was produced on the right hind limb,
Each animal was used once only, and was euthanized based on the method of Bennet & Xie (1988)17 and Attal
immediately after the experiments. All experiments et al. (1990).18 Then the animal's right sciatic nerve was
were carefully conducted according to the ethical exposed and freed from the surrounding tissues
guidelines for the use of experimental pain in conscious attachment, and a 2-3 mm long nerve segment was
animals put forth by the International Association for dissected. Only one ligature with fine metal wire was
the Study of Pain. secured around the nerve and the nerve was returned to
its bed.
Surgical procedure for nerve ligation.
The animals were anaesthetized with intraperitoneal Electrical stimulation of nerve
injection of sodium thiopental (40mg/kg). The unilateral This process started from the day of surgery. Under

Fig 2. Raw electromyograms (EMGs) of biceps femoris (left side) and gluteus maximus (right side) muscles after
electrical stimulation of sciatic nerve on the 3rd, 7th, 10th and 14th day in treated group after nerve ligation
in mice. Control EMG signals of biceps femoris and gluteus maximus muscles are shown in upper row.

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 Electrical stimulation of ligated sciatic nerve in mice
Eur J Transl Myol 27 (3): 173-182

Fig 3. Effect of electrical stimulation on Biceps femoris muscle force. The EMG signal was recorded on the 3rd,
7th, 10th, 14th day after ligation in non ES-Ligated group (o), ES-Ligated group (∆) and control group (∎).
Each point is mean ± SEM of Integral Absolute Value (IAV) for 10 mice. (**p<0.01, ***p<0.001 different
from respective control group, + ++P<0.001 different from respective non ES-Ligated group).

light anesthesia with ether, the sciatic nerve was re- drawing of the experimental set-up is shown in Figure 1.
exposed and gently suspended on a pair of stainless-
Treatment protocols
steel stimulatory electrodes. The proximal nerve stump
was then electrically stimulated for 40 seconds per day To study the effect of electrical stimulation (ES), mice
(20 pulses/ contraction). The stimulatory pulse were randomly divided into 3 groups of 10. Group 1:
(duration: 0.2 millisecon, frequency: 100 Hz, amplitude Ligated without electrical stimulation (non ES-Ligated).
15 milliAmpere) was delivered by a stimulator In this group sciatic nerve was ligated and allowed to
(Harvard, U.K) being connected to a voltage-to-current recover naturally. On the 3rd, 7th, 10th and 14th day,
convertor circuit using IC (LM 134, U.S.A). The surface EMG of BF and GS muscles were recorded in
amount of current delivered to each animal was this group. Group 2: Ligated with electrical stimulation
monitored by an oscilloscope (Tektronix, TDS 1002, (ES-Ligated) or treated group. In this group sciatic
TEXAS) to ensure that 15mA current was being nerve was ligated and it was under electrical stimulation
delivered. daily for a period of 2 weeks. The first ES was just after
the surgery. Surface EMG signals were recorded on the
Electromyography. 3rd, 7th, 10th and 14th day after nerve ligation. Group
Evoked surface EMG was recorded from Biceps 3: In this control group the sciatic nerve was intact and
Femoris (BF) and Gluteus Maximus (GM) muscles on EMG signals of BF and GS muscles were recorded.
the 3rd, 7th, 10th and 14th day after sciatic nerve ligation
Signal processing
on the right hind limb. To record, first the electrode sites
were shaved and cleaned with alcohol, and the lubricant The raw EMG data were stored and processed off line.
gel was applied for better conductivity. A pair of The digitized data were viewed before processing to
recording disposable adhesive Ag-Agcl electrodes reject those with insufficient signal level or with
(Biopac Company) placed on each end of the muscle artefacts. All signal processing was performed using
(totally 4 electrodes). The fifth electrode (reference) was custom programs written with LabView (version 6,
placed on the vertebral column; the electrodes were National Instruments, Austin, TX). To study muscle
taped securely to avoid excessive lead movements. Snap power or force, EMG signals were processed in the time
leads were used to connect the electrodes to the domain (Integral Absolute Value: IAV). In this method
amplifier (2 channels). All raw myoelectric signals were first signal was rectified and then root mean square
amplified (band width: 8-1600Hz). Evoked EMG was (RMS) was obtained and finally linear envelope was
recorded for 5 sec (5000 samples) during the final 10 determined in order to derive muscle power from the
sec of the sciatic nerve stimulation. EMG signals were EMG signal. The frequency domain of the surface EMG
digitized at 1KHZ with an A/D card (ADVENTECH, signals was used to study muscle sensitivity and the
818 PCL -818 HG). Data were collected and saved on a number of frequency components was considered as
personal computer for statistical analysis. The schematic muscle sensitivity index. Increased frequency
components of the EMG signal was interpreted as

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 Electrical stimulation of ligated sciatic nerve in mice
Eur J Transl Myol 27 (3): 173-182

Fig 4. Effect of electrical stimulation on Gluteus maximus muscle force. The EMG signal was recorded on the 3rd,
7th, 10th, 14th day after ligation in non ES- Ligated group (o), ES-Ligated group (∆) and control group (∎).
Each point is mean ±SEM of Integral Absolute Value (IAV) for 10 mice. (*** p<0.001 different from
respective control group).

muscle hyperalgesia or muscle hypersensitivity. First muscle force from the 7th day onward. Significant
Fast Fourier Transform (FFT) of signal was obtained, differences between the 7th and 10th day (p<0.05), 10th
rectified, filtered, analyzed and finally the number of and 14th day (p<0.001), 3rd and 10th day (p<0.001)
frequency component were counted using a peak were observed.
detector. A sample of raw EMG is shown in Figure 2.
Effect of sciatic nerve stimulation on the muscle force
Statistical analysis of Gluteus Maximus
Analysis of variance (ANOVA) followed by Tukey test The results are shown in Figure 4. There is a significant
was used to evaluate the significance of the results. All difference between experimental groups (ES-Ligated
data were expressed in terms of mean ± standard error and non ES-Ligated) as compared with control group
of mean (SEM), and p<0.05 was considered to be (p<0.001), but no significant difference was found in
significant. muscle force between the two experimental groups.
Further analysis showed that in ES-Ligated group there
Results
is a significant gradual increase in muscle force, i.e.,
Effect of sciatic nerve stimulation on the muscle force that there are significant differences between the 3rd and
of Biceps Femoris 7th day, 7th and 10th day , 10th and 14th day (p<0.05). In
The effect of electrical nerve stimulation on biceps non ES-Ligated group there is significant differences
femoris EMG signals is shown in Figure 3, in which between the 10th and 14th day (p<0.01), 3rd and 10th day
ES-Ligated (ligated with electrical stimulation or (p<0.01), and 7th and 14th day (p<0.01), but no
treated) and non ES-Ligated (ligated without electrical significant differences were found between the 3 rd and
stimulation) groups compared with each other and with 7th day or 7th and 10th day (p>0.05). No significant
control group. Analysis of variance revealed significant differences were found between the other groups
difference between ES-Ligated and non ES-Ligated Effect of sciatic nerve stimulation on the sensitivity of
groups (p<0.001). There is also a significant difference Biceps Femoris
between the two experimental groups versus control
The effect of ES on the number of frequency
group (p<0.001). Further analysis showed that there is
components of EMG signals of BF muscle is shown in
no significant difference between ES-Ligated and non
Figure 5.The results of ANOVA showed that there is a
ES-Ligated groups on the 3rd, 7th, 10th day after
significant difference between the two experimental
ligation. However, the main difference occurred on the
groups versus the control group (p<0.001), indicating
14th day after treatment with ES. A significant increase
that the number of frequency components is
in the EMG response and muscle force was also
significantly higher in experimental groups versus
obtained (p<0.001) and reaches to 80.9% of that of
control group. There is also significant difference
control group. Further analysis showed that in ES-
between ES-Ligated and non ES-Ligated groups
Ligated group, there is a significant gradual increase in

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Eur J Transl Myol 27 (3): 173-182

Fig 5. Effect of electrical stimulation on Biceps femoris muscle sensitivity. The EMG signal was recorded on the
3rd, 7th, 10th, 14th day after ligation in non ES-Ligated group (o), ES- Ligated group (∆) and control group
(∎). Each point is mean ± SEM of the frequency number for 10 mice. (*p<0.05, **p<0.01, ***p<0.001
different from respective control group, +++ p<0.001 different from respective non ES-Ligated group).

(p<0.001). Although no difference between these two evaluation of the repeatability of EMG variables is of
groups were found on the 14th day (p>0.05), on the 7th considerable relevance for the clinical use of this
day there was a significant difference between technique.23 In the present study, we have found a
experimental groups (p<0.001), thus the number of significant reduction in muscle power after sciatic nerve
frequency components is higher in ES-Ligated group. ligation. This finding confirms previous data that when
Further analysis between experimental groups showed muscles lose neural drive, they lose ability to generate
in non ES-Ligated group there is only significant force due to atrophy of their muscle fibers. 2,10,24,25 Our
difference between the 3rd and 14th day (p<0.05), results revealed that sciatic nerve electrical stimulation
however in ES-Ligated group there is significant produced a significant increase in the EMG response of
difference between the 3rd and 7th day (p<0.05). biceps femoris, indicating that its muscle strength
increased over time and reached 80.9% of that of
Effect of sciatic nerve stimulation on the sensitivity of normal muscle at the 14th day after sciatic nerve
Gluteus Maximus ligation, thus motor function was recovered better in
Figure 6 shows the comparison of the effect of ES on ES-treated mice. However, non-ES ligated (non-
the number of frequency components of GM muscle treated) group did not show significant increase in BF
among groups. Analysis of variance did not show muscle power on the 14th day. These results confirm Xu
significant difference in the number of frequency et al. (2014) study that recorded gastrocnemius muscle
components between non ES-Ligated with control group needle EMG, and reported nerve ES one day after
(p>0.05), or between ES-Ligated group with control surgery increases muscle action potential parameters
group (p>0.05). No difference was found between the such as motor nerve conduction velocity and peak
two experimental groups (p>0.05). amplitude and decrease latency onset of muscle action
potentials.27 It seems that ES induces structural changes
Discussion
in the muscle that, in turn, improves muscle function.
Surface electromyography (SEMG) is a non-invasive Our hypothesis confirms other studies indicating that
method for evaluation of muscle physiology. The time following nerve ES the number of myelinated fibers in
and frequency domains of the signal have been used to distal nerve stump,27 the number of regenerated axons,
examine central and peripheral aspects of the number of motoneurons and the thickness of myelin
neuromuscular physiologic function, as well as the sheath were significantly increased as compared to
motor unit activation modulate muscular force control group.28 In accordance with our results, there is
production.15,19,20 The EMG signal amplitude reflects evidence indicating that neuromuscular electrical
the number of activated motor units and their firing stimulation improved muscle strength.10 There is more
rates, and its frequency contents may be associated with evidence indicating that ES restores the paralyzed
the number of muscle fiber action potentials.21,22 Hence

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Fig 6. Effect of electrical stimulation on Gluteus Maximus muscle sensitivity. The EMG signal was recorded on
the 3rd, 7th, 10th, 14th day after ligation in non ES-Ligated group (o), ES-Ligated group (∆) and control
group (∎). Each point is mean ± SEM of the frequency number for 10 mice.

muscle mass,29 and evoked visible rhythmic muscle results, one may suggest that ES could function as a
contractions in these muscles.24 Even in old muscles, mechanism to bridge the gap between the nerve stumps
the electrical stimulation can generate tetanic electrically and improve muscle force. In other words,
contractions maintaining their mass and maximum the possibility may exists that the ES result in changes
force.25 Willand et al. (2015) reported that ES of muscle in the excitation-contraction coupling mechanism
following peripheral nerve injury accelerates muscle probably be mediated through muscle membrane ion
reinnervation and its functional behaviors.13 channels which can accelerate depolarization and results
The mechanisms by which the electrical stimulation synchronized activation of motor units and /or increase
exerts its effect are not clear. However, on the basis of of force generating capacity on the 14th day, which is in
available evidence we conclude that ES may improve accordance with finding of Ashley et al. (2007).33 Along
muscle function through the following mechanisms. with these results, it has been suggested that bridging is
Muscle power is a mechanical quantity which in our the best type of peripheral nerve repair.34 But what is the
experiments was evaluated by evoked EMG. When a molecular mechanism behind this bridging? Another
nerve is transected and sutured, axonal regeneration explanation for the effect of ES in our study could be
from the proximal stump into inappropriate distal that gene expression of nerve growth factors is
pathways after nerve lesion has been long recognized as increased. Nerve growth factor (NGF) is normally
a factor contributing to poor functional recovery. 30 produced in muscle on local injury and inflammation
Therefore innervation is a critical factor for the support and is known to play a role in regeneration after muscle
of the structural and functional integrity of skeletal injury.35 Denervation leads to increased NGF production
muscles.26 A possibility is that in our experiments by skeletal muscle,36 ‎and ES induces up-regualtion of
sciatic nerve ES increases the number of correct muscular neurotrophic factors.13,37 It has been shown
projections to the distal stump and to related muscles. that expression of proteins such as neurotrophins whose
This is consistent with the results of Al-Majed et al. release at the level of the muscle might play a key role
(2000) and Brushart et al. (2002) who found that, if the in determining the accuracy of reinnervation are
proximal stump of a cut nerve is stimulated at the time increased.1,38 On the other hand, NGF is involved in
of its surgical repair, the speed of reinnervation of target regulation of muscle strength.39,40 Barmptsioti et al.
muscles and sensory-motor precisionares were (2011) reported that NGF administration ensured a
improved.4,31 Electrical activity may influence the significant increase of average number of myelinated
axonal path finding of developing neurons,32 and axons per μm and lead to better EMG results.41 Dose
enhance the speed and accuracy of nerve regeneration.27 and duration of NGF administration are factors that
Therefore ES may be effective in promoting nerve determine the extent of recovery following peripheral
regeneration after peripheral nerve injury. From our nerve injury.42 Zhao et al. (2015) confirmed efficacy

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Eur J Transl Myol 27 (3): 173-182

and safety of nerve growth factor for the treatment of been shown NGF generates secondary hyperalgesia via
neurological diseases.43 Indeed, there is evidence a central mechanism.46 Our results have shown that
indicating that electrical muscle stimulation increases there was no significant difference in muscle sensitivity
expression of genes of motor endplate, calcium binding of GM between experimental groups with control or
proteins and acethylcholine receptors in muscle between experimental groups. Thus ligation could not
following spinal cord lesion.29 The present study induce muscle hyperalgesia in experimental groups.
revealed that ES did not affect GM muscle force. This This can be due to innervation of this muscle as
muscle seems to function differently from BF muscle. mentioned earlier or may be because of less production
This is probably because of its innervation, in fact BF of mediators involved in hyperalgesia. It seems that
and GM muscles are innervated by different nerves. BF both mechanisms may account for this response. Some
is a double muscle receiving 2 nerves supplies. The evidence showed that frequency of ES is an important
nerve to the short head of the BF comes from the factor that may determine electrical stimulation
common peroneal part of the sciatic nerve, while long effectiveness.51,52 In fact, a limitation of our study is that
head of the muscle is innervated by tibial branch of the we did not examined the effect of different frequencies.
sciatic nerve. Whereas, GM is innervated by inferior Further research is required to confirm the effectiveness
gluteal nerve.44 According to our data, the number of of the nerve electrical stimulation (without stimulation
frequency components of BF muscle that represents of muscle nociceptors and producing pain) for nerve
muscle sensitivity significantly increased in repair, and precise molecular mechanisms involved.
experimental groups, indicating both of them were On the other hand, our results support recent clinical
hyperalgesic as compared with control group, however application of Functional Electrical Stimulation (FES)
there is no difference between experimental groups on in Spinal Cord Injury (SCI) and aging patients.53-60
the 14th day. This increased EMG activity, muscle In conclusion, the results of this study have shown that
hyperalgesia, may be secondary (increased electrical stimulation of sciatic nerve accelerates
responsiveness to nociceptive stimuli outside the site of through local mechanisms sciatic nerve repair and
injury). Whether the hyperalgesia responses of biceps indirectly improves BF muscle force to a comparable
femoris muscle in the present study is mediated through level of the control without affecting muscle sensitivity.
a cutaneous or visceral mechanism should be clarified. ES of sciatic nerve, indeed, had no effect on force and
In the present study as can be shown in both ES-Ligated sensitivity of the GM muscle that is innervated by the
and non ES-Ligated groups, ligation can induce inferior gluteal nerve.
hyperalgesia. According to our data, ES-treated group
were becoming hyperalgesic from the 3rd day, and there List of acronyms
was a significant difference between treated and non- BF – Biceps Femoris
treated groups on the 7th day (Figure 5). There are data EMG - electromyography
from Hirayama et al. (2001) indicating that electrical ES – electrical stimulation
stimulus applied to the sciatic nerve in rats was high FES - Functional Electrical Stimulation
enough to activate C-fibers and nerve reflexively FFT - First Fast Fourier Transform
excited muscles.45 However, we do not know whether GM – Gluteus Maximus
pain existed on the 7th day of the experiment. IAV - Integral Absolute Value
Pharmacologic studies are needed to show if this effect RMS - root mean square
will be reverse by analgesic agents used clinically to SCI - Spinal Cord Injury
treat muscle pain. On the other hand, there is some SEM - standard error of mean
evidence indicating that intramuscular injection of NGF SEMG - Surface electromyography
induces hyperalgesia.46-48 Furthermore, NGF increases Author’s contributions
voltage-gated Na+ channels activity in excitable
cells.49,50 It has been reported that the activity of these Dr. F. Samiee conceived the work, performed the
channels is linked to neuronal excitability in chronic bibliographical search and drafted the manuscript. Dr.
pain states.49 As we mentioned before; ES increases M.-R. Zarrindast approved the research and the final
NGF production. So, we hypothesize that NGF manuscript.
production is at its maximum level of production at day Acknowledgments
7 after ES which result in significant difference between
The technical assistance of Mr. Hamidreza Taghavi is
ES-ligated and non ES-ligated mice. This is consistent
gratefully acknowledged.
with the results of Brushart et al. (2002) who found that
the use of ES significantly increased the number of Ethical Publication Statement
axons crossing at the 4 and 7th day, with only a few We confirm that we have read the Journal’s position on
crossing after 2 weeks.31 It is possible that after this day issues involved in ethical publication and affirm that
a form of muscle adaptive behavior occurs, indicating this report is consistent with those guidelines.
repetitive input results in a negative feedback, and the
muscle sensitivity return to non-treated level. It has

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 Electrical stimulation of ligated sciatic nerve in mice
Eur J Transl Myol 27 (3): 173-182

Conflict of Interest 11. Chen TY, Yang YC, Sha YN, et al. Far-Infrared
None of the authors have conflicts of interests. Therapy Promotes Nerve Repair following End-to-
End Neurorrhaphy in Rat Models of Sciatic Nerve
Corresponding Author Injury. Evid Based Complement Alternat Med
Farzaneh Samiee, Biomedical Engineering Faculty, 2015;2015:207245. doi: 10.1155/2015/207245.
Science and Research Branch, Islamic Azad University, 12. Willand MP, Holmes M, Bain JR., et al.
Tehran, Iran. Phone:+98912 1967190 Determining the effects of electrical stimulation on
E-mail: [email protected] functional recovery of denervated rat
gastrocnemius muscle using motor unit number
E-mails of co-author estimation. In Annual International Conference of
ZarrindastMohammad-Reza: [email protected] the IEEE Engineering in Medicine and Biology
Society 2011; (pp. 1977-1980).
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