|Year : 2015 | Volume
| Issue : 3 | Page : 742-747
Effect of electrical stimulation and stem cells on experimentally induced peripheral nerve injury in rats
Fawzy A Ashour1, Amani A Elbaz2, Nagwan A Sabek3, Suzan M Hazzaa4, Ebtehal M Metwally MSc 4
1 Department of Physiology, Faculty of Medicine, Al Azhar University, Cairo, Egypt
2 Department of Physiology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
3 Department of Biochemistry, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
4 Department of Physiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||13-Aug-2014|
|Date of Acceptance||19-Sep-2014|
|Date of Web Publication||22-Oct-2015|
Ebtehal M Metwally
Department of Physiology, Faculty of Medicine, Menoufia University, Quesna, 32631 Menoufia
Source of Support: None, Conflict of Interest: None
This work aimed to study the effect of low intensity electrical stimulation and Mesenchymal stem cells transplantation on repair of sciatic nerve crush injury.
Injury of peripheral nerves results in temporary or life-long neuronal dysfunction that can subsequently lead to economic or social disability. Despite early diagnosis and use of modern surgical techniques, functional recovery can never reach the pre-injury level. Several alternate approaches have been proposed to get beneficial effects on peripheral nerve regeneration, including application of electric field, transplantation of stem cells, and administration of neurotrophic factors.
Materials and Methods
48 albino rats weighing 180:250 gm were used in this study. Rats were divided into four equal groups (12 rats each): Sham surgery group: sciatic nerve was exposed but not crushed. Injured sciatic nerve control group: sciatic nerve was exposed and crushed. Mesenchymal stem cells (MSCs) transplantation group: Sciatic nerve injury was done, followed by Transplantation of (3 ΄ 10 5 cells/rat) mesenchymal stem cells intra-lesion immediately after injury. Electrical stimulation (ES) group: sciatic nerve injury was followed by electrical stimulation for 30 minutes. All procedures were followed by wound closure and post-surgical care. Serum malondialdehyde and total antioxidant capacity were estimated 48 hours after injury then electrophysiological studies were measured 8 weeks after injury.
Treatment with either ES or MSCs transplantation could accelerate and promote sciatic nerve functional regeneration over 8 weeks.
We concluded that both ES and MSCs transplantation improve peripheral nerve functional regeneration following crush nerve injury. Such effect makes those treatments beneficial for accelerating and giving better outcome of peripheral nerve functional regeneration.
Keywords: Electrical stimulation, peripheral nerve injury, stem cells
|How to cite this article:|
Ashour FA, Elbaz AA, Sabek NA, Hazzaa SM, Metwally EM. Effect of electrical stimulation and stem cells on experimentally induced peripheral nerve injury in rats. Menoufia Med J 2015;28:742-7
|How to cite this URL:|
Ashour FA, Elbaz AA, Sabek NA, Hazzaa SM, Metwally EM. Effect of electrical stimulation and stem cells on experimentally induced peripheral nerve injury in rats. Menoufia Med J [serial online] 2015 [cited 2022 Nov 29];28:742-7. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/742/167896
| Introduction|| |
Peripheral nerve transection or crush leads to acute myelinoaxonal degeneration in the distal area of the damaged nerve, called Wallerian degeneration . Different types of lesions have different prognosis . Crush lesions maintain the basal lamina, generating appropriate environment for regeneration, which is not observed in a transection injury . Despite early diagnosis and use of modern surgical techniques, functional recovery can never reach the preinjury level; this poor outcome may result from many factors, intrinsic and extrinsic to the nervous system . Approaches that promote functional recovery in peripheral nerve injury include cell therapy, neuromodulation [e.g. electrical stimulation (ES)], and application of neurotrophic factors . Cell transplantation has been proposed as a method of improving peripheral nerve regeneration .
In-vivo studies have shown that mesenchymal stem cells (MSCs) can improve nerve regeneration, by differentiating into Schwann-like cells, which support nerve fiber growth and myelination . Therefore, MSCs were chosen to promote simultaneous growth and differentiation of nerve fibers, blood vessels, and the supportive connective tissue . Many studies have investigated the influence of electrical fields on peripheral nerve regeneration using laboratory animals . Low-intensity ES was tested as an adjuvant to peripheral nerve regeneration as early as 1982 . It was recently reported that 30 min of low-intensity ES applied after injury can improve regeneration in crushed rat sciatic nerve .
The aim of our work was to study the effect of treatment with MSCs transplantation and ES on repair of sciatic nerve crush injury.
| Materials and methods|| |
A total of 48 albino rats weighing 180-250 g were used in this study. Animals were fed with standard laboratory chow and water, housed in animal house at faculty of Medicine Menoufia University under artificial light/dark cycle of 12 h. The animals were acclimatized to these conditions for 14 days before the experiment. They were divided into four equal groups (12 rats each): the Sham surgery group, in this group the sciatic nerve was exposed but not crushed; the injured sciatic nerve control group, in this group sciatic nerve was exposed and crushed; the MSCs transplantation group, in this group sciatic nerve injury was followed by transplantation of 3 × 10 5 cells/rat  MSCs, which were injected intralesion immediately after injury; and the ES group, in this group sciatic nerve injury was followed by ES by applying the electrodes 5 mm proximal to the injured site, using a biphasic current pulse (100 μs pulse width, 20 Hz pulse rate, 2 mA amplitude) for 30 min. The surgical wound was kept moist throughout the stimulation period by covering it with wet sterile gauze . Procedures of all groups were followed by wound closure and postsurgical care for 8 weeks.
This experiment was approved by the Research Ethics Committee at Faculty of Medicine, Menoufia University. Human umbilical cord blood (hUCB) was collected from full-term pregnant women after taking written consents. Thereafter, samples were prepared for tissue culture for preparation of MSCs. The cells were gated out by positive expression of CD105 and negative expression of CD34 and CD45.
Mesenchymal stem cells preparation
hUCB was collected from normal volunteers using strict aseptic techniques. Tissue culture plastic flasks 25 cm2 were prepared for culture by adding a minimum essential medium supplemented with 20% fetal bovine serum, 1% antibiotic/antimycotic, and 1% glutamine. Nonadherent cells were removed and fresh medium was added to the culture flask. Cellular growth was assessed daily under inverted microscope. When the cells reached 50-60% confluence, they were harvested after trypsin/ethylenediaminetetraacetic acid (0.025%).
Method of sciatic nerve injury
Rats were anesthetized with pentobarbital (40 mg/kg intraperitoneal) and allowed to recover for 8 weeks after surgery . Sciatic nerve was exposed and crushed with 3 mm wide hemostat for 1 min . It was applied 10 mm proximal to sciatic trifurcation then the wound was closed and further antisepsis was added .
Fasting blood samples were collected from retro-orbital venous plexus of rats, using fine nonheparinized capillary tubes introduced into the medial epicanthus of rat's eye. Two milliliters of blood were collected and centrifuged in a clean graduated tube at 3000 rpm for 5 min (Narco-Biosystem, UK). The supernatant serum was collected in a dry tube for estimation of malondialdehyde (MDA) and total antioxidant capacity (TAC). Colorimetric estimation of MDA was performed using thiobarbituric acid reactive substance for measuring the peroxidation of fatty acids as oxidative stress marker ,. The assay of TAC was performed by allowing the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide (H 2 O 2 ). The antioxidants in the sample eliminate a certain amount of the provided H 2 O 2 . The residual H 2 O 2 was determined colorimetrically .
These were performed at the end of the eighth postsurgical week.
Rats were anesthetized using pentobarbital. Two stimulating hooked electrodes were placed around the sciatic nerve 5-mm proximal to the crush site. Electrical current application initiated with monophasic, single, square pulse with a duration of 1 ms and an intensity of 10 mA produced by an electric stimulator (EMG100C; Biopac Systems Inc., CA, USA). The intensity was gradually increased until the supramaximal stimulation that ensured maximal amplitude was reached (1 mA). Thereafter, the recorded signals were digitally converted with an MP 150 (Biopac Systems Inc.). The latency period and amplitude were measured. The latency was measured from the stimulus to the takeoff of the first negative deflection and the amplitude was calculated from the baseline to the maximal negative peak . A heating lamp was used to keep rat's body temperature at ~37°C during the tests .
Method of nerve conduction velocity
Animals were anesthetized with pentobarbital, and then were killed by cervical dislocation. Left sciatic nerves were dissected from the spinal emergence to the knee and stored in normal Ringer's solution. Nerve stimulation and recording was accomplished using Biopac MP 150 data acquisition system. A stimulus was applied at 50 μs duration, with intensity set at ~1.25 times, which gave the maximum height of the compound action potential. Nerve conduction velocity (NCV) was measured by dividing the distance between the stimulating and recording electrodes by the time elapsed between the initiation of the stimulus and the time when 50% of the increase of the component of compound action potential was reached.
The SPSS (version 16; SPSS Inc., Chicago, Illinois, USA) statistical tool was used for analysis of data. The results of experiment were expressed as mean ± SEM. The significance of differences between groups was determined by one-way analysis of variance and Student's t-test. The significance of differences was determined at P value less than 0.05 .
| Results|| |
[Table 1] shows values of NCV (mm/s) in all groups at the end of the eighth postsurgical week. The table shows the mean value ± SEM of NCV in the injured control group (26.71 ± 1.3), which was significantly lower than the corresponding value in the sham surgery group (39.08 ± 1.31) (P < 0.001). The same table shows that the mean value ± SEM of NCV in the MSCs transplantation group and ES group was 34.87 ± 1.3 and 34.96 ± 1.29, respectively, which was significantly high (P < 0.01) when compared with NCV in the injured control group (26.71 ± 1.3), but there was no significant difference (P > 0.05) when compared with the sham surgery group NCV (39.08 ± 1.31).
|Table 1: Nerve conduction velocity (mm/s) at the end of the eighth postsurgical week|
Click here to view
[Figure 1] and [Figure 2] illustrate values of electromyography (EMG) amplitude (mv) and latency (s) in all groups at the end of the eighth postsurgical week. As shown in the table, the mean value ± SEM of EMG amplitude in the injured control group was 0.70 ± 0.049, which was significantly low (P < 0.001) when compared with the corresponding value in the sham surgery group (1.84 ± 0.066). In addition, EMG amplitude in the MSCs transplantation group and ES group was 1.52 ± 0.149 and 1.53 ± 0.108, respectively, which was significantly high (P < 0.001) when compared with the corresponding value in the injured control group (0.70 ± 0.049), but it showed no significant difference (P > 0.05) when compared with the sham surgery group (1.84 + 0.066).
|Figure 1: The curves of the electromyography in (a) the sham surgery group, (b) the injured control group, (c) the mesenchymal stem cells (MSCs) transplantation group, and (d) the electrical sti mulation (ES) group.|
Click here to view
|Figure 2: The electromyography (EMG) amplitude and EMG la tency in all groups.|
Click here to view
Regarding the EMG latency(s) in all groups at the end of the eighth postsurgical week, the mean value ± SEM of EMG latency was 1.003 ± 0.031 in the injured control group, which was significantly high (P < 0.001) when compared with the corresponding value in the sham surgery group (0.797 ± 0.002). In addition, the EMG latency in the MSCs transplantation group and ES group was 0.812 ± 0 and 0.811 ± 0.001, respectively, which was significantly low (P < 0.001) when compared with the corresponding value in the injured control group (1.003 ± 0.031), but there was no significant difference (P > 0.05) when compared with the sham surgery group (0.797 ± 0.002).
[Table 2] shows the levels of serum MDA (nmol/ml) and serum TAC (mmol/l) in all groups. As shown in the table, the mean value ± SEM of serum MDA level in the injured control group was 10.76 ± 0.36 nmol/ml, which was significantly higher (P < 0.001) than the corresponding value in the sham surgery group (3.87 ± 0.12 nmol/ml). In addition, it shows the mean value ± SEM of the serum MDA levels of the MSCs transplantation and ES groups (5.88 ± 0.1 and 6.04 ± 0.14, respectively), which was significantly low (P < 0.001) when compared with the corresponding value in the injured control group (10.76 ± 0.36).
|Table 2: Serum malondialdehyde (nmol/ml) and serum total antioxidant capacity (mmol/l) in all groups|
Click here to view
The same table shows the mean value ± SEM of serum TAC in the injured control group (0.73 ± 0.040), which was significantly low (P < 0.001) when compared with the sham surgery group (2.56 ± 0.044). However, the mean values ± SEM of serum TAC in the MSCs transplantation and ES groups were 1.64 ± 0.019 and 1.62 ± 0.026, respectively, showing significantly higher values (P < 0.001) when compared with corresponding value (0.73 ± 0.040) in the injured control group.
| Discussion|| |
In the present study, the crush nerve injury model produced an axonotmetic peripheral nerve injury. The electrophysiological studies in the injured control group after 8 weeks of injury revealed that NCV and EMG amplitudes were significantly low, whereas EMG latency showed significant prolongation when compared with corresponding values in the sham surgery group. Deterioration of the electrophysiology of the crushed sciatic nerve in our study may be explained by direct effect of trauma . In addition, it may be due to crush-related ischemia or reperfusion injury, which increases neural damage, or due to activation of reactive oxygen molecules . Supporting this study, we detected significantly elevated serum MDA levels and decreased serum TAC in the injured control group when compared with the sham surgery group after 48 h of injury.
It has been consistently reported that MDA levels remained high initially and then decreased in ischemia-reperfusion injury of sciatic nerve in rats . The peripheral nervous system, similar to the central nervous system, has a high level of myelin and polyunsaturated lipids, which make it more susceptible to free oxygen radical-mediated lipid peroxidation. As a consequence, free radicals attack the lipid membranes, causing neural disintegration and degeneration . In addition, the ability of injured peripheral axons to regenerate is generally slow; this could be due to a decline in neurotrophic support in the tissue surrounding the regenerating axons over time .
Electrophysiological studies in the ES group showed marked improvement when compared with the injured control group at the end of the eighth postsurgical week revealed by higher NCV and EMG amplitude and lower EMG latency, which were significant when compared with corresponding values in the injured control group. At the same time, the previous values were insignificant when compared with the corresponding values in the sham surgery group indicating improved sciatic nerve regeneration. The improved nerve functional regeneration can be explained by the ameliorative effect of ES on oxidative stress of the crush nerve injury revealed by significantly decreased serum MDA levels and elevated serum TAC in the ES group when compared with the injured control group after 48 h of injury. Sayyed et al.  reported that vagus nerve ES resulted in significant decreased lipid peroxidation and increased total thiols in the treated animals of cerebral ischemia and reperfusion model. Total thiols are involved in many biological activities including neutralization of reactive oxygen species .
Alrashdan et al.  reported that the same protocol of ES can enhance axonal regeneration when applied immediately after nerve crush injury; the same study showed that quantification of brain derived neurotrophic factor (BDNF) levels in dorsal root ganglion sensory neurons by means of real-time PCR showed significantly higher levels in the ES group. It may be due to direct role of neurotrophins (especially BDNF) in maintaining the viability of injured neurons, together with upregulation of high-affinity receptors such as tropomyosin-related kinase after axotomy . Activation of l-type voltage-sensitive Ca 2+ channels or the non-N-methyl-d-aspartate subtype of glutamate receptor leads to an enhancement of BDNF mRNA levels in hippocampal neurons and in cortical neurons . Tyreman et al.  reported that, when endogenous BDNF was blocked by a functional blocking antibody administered during the first 3 days postinjury, the augmenting effects of ES on axon regeneration were abolished.
Electrophysiological studies in the MSCs transplantation group showed marked improvement when compared with the injured control group at the end of the eighth postsurgical week revealed by higher NCV and EMG amplitude and lower EMG latency, which were significant when compared with corresponding values in the injured control group. At the same time, the previous values were insignificant when compared with the corresponding values in the sham surgery group indicating improved sciatic nerve regeneration. The improved nerve regeneration can be explained by the ameliorative effect of MSCs transplantation on oxidative stress of the crush nerve injury, revealed by significantly decreased serum MDA levels and elevated serum TAC in the MSCs transplantation group when compared with the injured control group after 48 h of injury and improved neurotrophic support in addition to reported ability of undifferentiated stem cells to differentiate into Schwann cells in vivo.
Pang et al.  reported that hUCB MSCs enhanced peripheral nerve regeneration functionally, electrophysiologically, and their results suggested that undifferentiated stem cells can differentiate into Schwann cells in vivo. According to Cuevas et al. , 5% of transplanted stem cells become Schwann cells. Stem cells transplanted into lesions in the central nervous system could differentiate into oligodendrocytes and astrocytes. These cells then integrated into the axonal pathways that can regenerate and remyelinate the injured axons .
Several in-vitro and in-vivo studies proved that MSCs can potentially regulate the redox environment. Iyer et al.  found that BM-MSCs can maintain the steady-state of cysteine and glutathione in plasma during endotoxemia and reduce the oxidation of the cysteine and glutathione redox system. hMSCs possess the main enzymatic and nonenzymatic mechanisms to detoxify reactive species and to correct oxidative damage . Sun et al.  confirmed that the antioxidation effect of adipose tissue-derived MSCs play an important role in ameliorating lung ischemia-reperfusion injuries.
| Conclusion|| |
We concluded that both MSCs transplantation and our protocol of ES improved peripheral nerve functional regeneration. This can be revealed by improved electrophysiological results of both groups, which may be explained by decreased oxidative stress.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rotshenker S. Wallerian degeneration: the innate-immune response to traumatic nerve injury. J Neuroinflammation 2011; 8
Baptista AF, Gomes JRS, Oliveira JT, Santos SMG, Vannier-Santos MA, Martinez AMB. High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse. J Peripher Nerv Syst 2008; 13
Gao S, Fei M, Cheng C, Yu X, Chen M, Shi S, et al.
Spatiotemporal expression of PSD-95 and nNOS after rat sciatic nerve injury. Neurochem Res 2008; 33
Pan HC, Chin CS, Yang DY, Ho SP, Chen CJ, Hwang SM, et al.
Human amniotic fluid mesenchymal stem cells in combination with hyperbaric oxygen augment peripheral nerve regeneration. Neurochem Res 2009; 34
Sung MS, Jung HJ, Lee JW, Lee JY, Pang KM, Yoo SB, et al.
Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves. Neural Regen Res 2012; 26
Cuevas P, Carceller F, Dujovny M, Garcia-Gomez I, Cuevas B, Gonzalez-Corrochano R, et al.
Peripheral nerve regeneration by bone marrow stromal cells. Neurol Res 2002; 24
Choi BH, Zhu SJ, Kim BY, Huh JY, Lee SH, Jung JH. Transplantation of cultured bone marrow stromal cells to improve peripheral nerve regeneration. Int J Oral Maxillofac Surg 2005; 34
Lopes FRP, Campos LCD, Correa JD, Balduino A, Langone F, Borojevic R, et al.
Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice. Exp Neurol 2006; 198
Alrashdan MS, Sung ME, Kwon YK, Chung HJ, Kim SJ, Lee JH. Effects of combining electrical stimulation with BDNF gene transfer on the regeneration of crushed rat sciatic nerve. Acta Neurochir 2011; 153
Alrashdan MS, Park JC, Sung MA, Yoo SB, Jahng JW, Lee TH, et al.
Thirty minutes of low intensity electrical stimulation promotes nerve regeneration after sciatic nerve crush injury in a rat model. Acta Neurol Belg 2010; 110
Joghataei MT, Bakhtiari M, Pourheydar B, Mehdizadeh M, Faghihi A, Mehraein F, et al.
Co-transplantation of Schwann and bone marrow stromal cells promotes locomotor recovery in the rat contusion model of spinal cord injury. Yakhteh Medical Journal 2009; 12
Wan L, Zhang S, Xia R, Ding W. Short-term low-frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain-derived neurotrophic factor on Schwann cell polarization. J Neurosci Res 2010; 88
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978; 90
Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Ann Biochem 1979; 95
Koracevic D, Koracevic G. Measurement of total antioxidants. J Clin Pathol 2001; 54
Lu MC, Ho CY, Hsu SF, Lee HC, Lin JH, Yao CH, et al.
Effects of electrical stimulation at different frequencies on regeneration of transected peripheral nerve. Neurorehabil Neural Repair 2008; 22
Ferguson GA, George A, Takane Y, et al.
Statistical analysis in psychology and education. 6th ed. Montréal, Quebec: McGraw-Hill Ryerson Limited; 2005. 333-360.
Emel E, Ergun SS, Kotan D, Gursoy EB, Parman Y, Zengin A, et al.
Effects of insulin-like growth factor-I and platelet-rich plasma on sciatic nerve crush injury in a rat model. J Neurosurg 2011; 114
Wong KH, Naidu M, David P, Abdulla MA, Abdullah N, Kuppusamy UR, et al.
Peripheral nerve regeneration following crush injury to rat peroneal nerve by aqueous extract of medicinal mushroom Hericium erinaceus
(Bull.: Fr) Pers. (Aphyllophoromycetideae). Evid Based Compl Altern Med 2010; 2011
Bagdatoglu C, Sarray A, Surucu HS, Ozturk H, Tamer L. Effect of trapidil in ischemia/reperfusion injury of peripheral nerves. Neurosurgery 2002; 51
Gudemez E, Ozer K, Cunningham B, Siemionow K, Browne E, Siemionow M. Dehydroepiandrosterone as an enhancer of functional recovery following crush injury to rat sciatic nerve. Microsurgery 2002; 22
Gordon T. The role of neurotrophic factors in nerve regeneration. Neurosurg Focus 2009; 26
Sayyed HG, Idriss NK, Darwish AM. Effect of vagus nerve stimulation on focal transient cerebral ischemia and reperfusion in adult male white New Zealand rabbits. Ibnosina J Med Biomed Sci 2013; 2
Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med 1999; 27
Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME. Ca 2
influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 1998; 20
Tyreman JG, Pettersson LME, Verge VM, Gordon T. BDNF-mediated acceleration of motor axonal regeneration by brief low frequency electrical stimulation (ES). Soc Neurosci 2008; 33
Pang KM, Sung MA, Alrashdan MS, Yoo SB, Jabaiti S, Kim SM, et al.
Transplantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration. Neural Regen Res 2010; 5
Vroemen M, Aigner L, Winkler J. Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways. Eur J Neurosci 2003; 18
Iyer SS, Torres-Gonzalez E, Neujahr DC, Kwon M, Brigham KL, Jones DP, et al.
Effect of bone marrow-derived mesenchymal stem cells on endotoxin-induced oxidation of plasma cysteine and glutathione in mice. Stem Cells Int 2010; 868076.
Valle-Prieto A, Conget PA. Human mesenchymal stem cells efficiently manage oxidative stress. Stem Cells Dev 2010; 19
Sun CK, Yen CH, Lin YC, Tsai TH, Chang LT, Kao YH, et al.
Autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model. J Transl Med 2011; 9
[Figure 1], [Figure 2]
[Table 1], [Table 2]