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ORIGINAL ARTICLE
Year : 2015  |  Volume : 28  |  Issue : 3  |  Page : 737-741

Effect of Schwann and mesenchymal stem cells on experimentally induced sciatic nerve injury in rats


1 Department of Physiology, Faculty of Medicine, Al-Azhar University, Shebeen Elkom, Menoufia, Cairo, Egypt
2 Department of Physiology, Faculty of Medicine, Suez Canal University, Shebeen Elkom, Menoufia, Cairo, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Shebeen Elkom, Menoufia, Cairo, Egypt
4 Department of Clinical Physiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission10-Aug-2014
Date of Acceptance13-Oct-2014
Date of Web Publication22-Oct-2015

Correspondence Address:
Reda A Abo Elsoud
Department of Clinical Physiology, Faculty of Medicine, Menoufia University, El Shouhada, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.165827

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  Abstract 

Objective
Transplantation of bone marrow stromal cells (BMSCs) and Schwann cells (SCs) can facilitate axon regeneration in peripheral nerve injuries. The aim of this study was to explore the effect of transplantation of BMSCs and SCs on electrophysiological recording after injury of the sciatic nerve in the rat.
Materials and methods
In this study, 40 adult male albino rats (250-300 g) were used. BMSCs and SCs were cultured. Rats were divided randomly into four equal groups: group 1, control without nerve injury; group 2, nerve injury without cell transplantation; group 3, nerve injury with BMSCs transplantation; and group 4, nerve injury with SCs transplantation. Standardized crush injury of the sciatic nerve (axonotmesis) was performed by surgical hemostat at the first lock for 1 min; BMSCs and SCs were separately transplanted intralesionally. After 8 weeks, electrophysiological recordings were analyzed by one-way analysis of variance.
Results
Electrophysiological analysis showed a significant improvement in the cell transplantation groups compared with the injured group (P < 0.01).
Conclusion
BMSCs and SCs may potentially enable recovery after a standardized injury to the sciatic nerve in rats (axonotmesis). Electrophysiological evaluation confirms this improvement after transplantation of SCs and BMSCs, with no significant difference between them.

Keywords: Bone marrow stromal cells, peripheral nerve, regeneration, Schwann cells, transplantation


How to cite this article:
Ashour FA, Elbaz AA, Attia FM, El-Kotb SM, Abo Elsoud RA. Effect of Schwann and mesenchymal stem cells on experimentally induced sciatic nerve injury in rats. Menoufia Med J 2015;28:737-41

How to cite this URL:
Ashour FA, Elbaz AA, Attia FM, El-Kotb SM, Abo Elsoud RA. Effect of Schwann and mesenchymal stem cells on experimentally induced sciatic nerve injury in rats. Menoufia Med J [serial online] 2015 [cited 2020 Feb 23];28:737-41. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/737/165827


  Introduction Top


Peripheral nerve injuries are injuries that cause loss of sensory and motor function. These injuries are most common in young adults and are often caused by traffic accidents, occupational trauma with machinery knifes and glass, and self-destructive behavior [1]. Different procedures have been used to improve regeneration of peripheral nerves; cell transplantation is one of the cell therapy and tissue engineering strategies aimed at the creation of a favorable microenvironment for tissue regeneration [2].

Bone marrow stromal cells (BMSCs) have the ability to differentiate into different cell lines such as osteoblasts, cartilage, fat, ligament, muscle, and neurons [3]. BMSCs have the ability to release neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor (VEGF), as well as to produce extracellular matrix proteins such as collagen I, collagen IV, fibronectin, and laminin; there is good evidence to support the hypothesis that transplantation of BMSCs may repair peripheral nerve injuries [4]. Also, they can transdifferentiate into Schwann cells (SCs)-like cells, which lead to nerve regeneration when transplanted to the site of the sciatic nerve injury [5].

Another cell type used for the repair of peripheral nerve injures is the SCs. SCs and their basal lamina are crucial components in the environment through which regenerating axons grow to reach their peripheral targets. SCs can myelinate and produce physical support for axonal growth when they are injured [6]. SCs produce neurotrophic factors, extracellular matrix molecules, and associated integrins that promote the growth of the nerve [7]. Wallerian degeneration activates the SCs and they supply ideal trophic substances for axonal regeneration by secreting growth factors such as NGF, hepatocyte growth factor (HGF), VEGF, and BDNF [8]. This work aimed to assess the role of transplantation of SCs and bone marrow mesenchymal stem cells (MSCs) in sciatic nerve regeneration in albino rats.


  Materials and methods Top


Experimental animals

Forty adult male albino rats, of local strains, weighing 200-250 g each were used in this investigation. Rats were purchased from the Military Animals Farm (Cairo). Rats were housed in a fully ventilated cages (six per cage) with free access to water and a semisynthetic balanced diet.

Rats were divided into the following groups:

  1. Group I: The control intact sciatic nerve group with skin injury (n = 10 rats), followed by wound closure with postsurgical care.
  2. Group II: The injured left sciatic nerve group (n = 10 rats), treated with physiological normal saline intralesion in the acute phase of injury, followed by wound closure with postsurgical care.
  3. Group III: Injured left sciatic nerve group (n = 10 rats), treated with 3×10 5 MSCs in physiological normal saline intralesion in the acute phase of injury, followed by wound closure with postsurgical care [9].
  4. Group IV: Injured left sciatic nerve group (n = 10 rats), treated with 3×10 5 SCs in physiological normal saline intralesion in the acute phase of injury, followed by wound closure with postsurgical care [9].


Methods of sciatic nerve injury

Standardized crush injury of the sciatic nerve

Animals were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal). When the sciatic nerve was exposed and detached from the surrounding tissues, a standard surgical hemostat was used to create a crush injury at a distance of 10 mm proximal to the trifurcation. A defect of 3 mm length was produced [10]. The sciatic nerve was crushed with a 3-mm-wide hemostat at the first lock for 1 min [11]. Immediately after the creation of the 3-mm-wide crush injury, SCs and BMSCs were injected directly without a scaffold at a density of 300 000 cells in physiological normal saline into the injured sciatic nerve using an insulin syringe with a 28-G needle. The rats in the sham control group received physiological normal saline without cells; then, the wound was closed in a single layer using 4-0 nylon sutures and further antisepsis with a povidone-iodine solution was applied [9].

Mesenchymal stem cells isolation [12]

The rat bone marrow was washed with PBS and seeded in 35 mm tissue culture plates in a density of 10 6 /plate and 8 ml complete medium [10% FBS, 1% antibiotic (penicillin/streptomycin)]. The cells were incubated in a humidified atmosphere of 5% CO 2 at 37C°. Twenty-four hours after seeding, nonattached cells were removed and the medium was exchanged with fresh complete medium. The culture was monitored on a daily basis and medium was exchanged every 5 days. The attached fibroblastoid cells recovered were trypsinized by trypsin/EDTA and passaged.

Schwann cells' isolation

SCs were isolated and obtained from the sciatic nerve of rats by removing the epineurium from the nerve by pulling out the individual nerve fascicles using fine sterile forceps, incubated in an enzymatic mixture containing hyaluronidase type I-s and collagenase type I, resuspended in culture medium, and then a MACS (Militenyi Biotech, GmbH Bergisch Gladbach, Germany) MicroBeads selection of SCs was performed on the basis of their surface expression of p75 [13].

Electrophysiological evaluation

Electromyography method

At the end of the 8 weeks postoperatively, rats from each group were anesthetized using the same mixture as for previous operations. Two stimulating hooked platinum bipolar electrodes were placed around the sciatic nerve 5-mm proximal to the transection site. Electrical current application was started with a 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., California, USA). The intensity was increased gradually until the supramaximal stimulation that ensured maximal amplitude was reached (1 mA). Thereafter, the recorded signals were digitally converted using an mp150 (Biopac Systems Inc.).

Recordings were made on a two-channel electromyography (EMG) system with the high-frequency filter set at 5 kHz and the low-frequency filter set at 1 Hz, a gain of 5000 Hz. A percutaneous puncture, ipsilateral to the surgical procedure + in the muscle origin - in the insertion and ground electrode into the rat's tail. The latency period and amplitude were measured. The latency was measured from stimulus to the take-off of the first negative deflection. The amplitudes from the baseline to the maximal negative peak were calculated [14].

Methods of nerve conduction velocity

Animals were anesthetized with pentobarbital sodium (100 mg/kg, intraperitoneal). Rats were killed by cervical dislocation and exsanguination.

Left sciatic nerves were dissected from the spinal emergence to the knee and stored in normal Ringer's solution. Nerve stimulation and recording were carried out using the Biopac mp150 data acquisition system. The nerve stimulation holder (8 × 4.5 × 2.5 cm) was acrylic and contained three chambers. All chambers were filled with Ringer's solution; the 15-35-mm segment of the nerve placed in the chamber such that it had good electrical contact with the measuring and stimulating electrodes. The temperature of Ringer's solution was monitored and maintained at room temperature (~20-23°C). A stimulus was applied at 50 μm/sec duration, with the intensity set at ~1.25 times, which provides 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 a stimulus artifact and the initiation of the stimulus.


  Results Top


Nerve conduction velocity result

NCV was determined for all groups 8 weeks after injury using the Biopac Acknowledge system.

[Table 1] and [Figure 1] show a statistically significant (P < 0.01) decrease in the NCV of the injured nontreated group (26.7 ± 1.3) compared with the normal group (39.08 ± 1.31), and a statistically significant increase in the NCV value (P < 0.01) in the MSC-treated group (34.92 ± 1.29) and SC-treated group (36.95 ± 2.27) compared with the injured nontreated group. There were no statistically significant differences (P > 0.05) in the MSC-treated and SC-treated groups.
Figure 1: Nerve conduction velocity (NCV) values in mm/s in the normal, injured nontreated, and Schwann-treated, and mesenchymal stem cell-treated group.

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Table 1: Nerve conduction velocity values in mm/s in normal, injured nontreated, Schwann cells-treated, and mesenchymal stem cell-treated groups

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Electromyography result

EMG was performed for all groups 8 weeks after injury using the Biopac Acknowledge system.

[Table 2] and [Figure 2] show a statistically significant (P < 0.01) decrease in the EMG amplitude values in the injured nontreated group (0.703 ± 0.049) compared with the normal group (1.83 ± 0.065) and a statistically significant increase in the EMG amplitude values (P < 0.01) in the MSC-treated group (1.466 ± 0.1286) and SC-treated group (1.49 ± 0.136) compared with the injured nontreated group. There were no statistically significant differences (P > 0.05) in the MSC-treated and SC-treated groups.
Figure 2: MAP amplitude in mV & latency in S in normal, injured non treated, mesenchymal stem cells & schwann cells treated group *significant when compared with the injured group.

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Table 2: Electromyography amplitude in mV and latency in S in normal, injured nontreated, mesenchymal stem cell- treated, and Schwann cell-treated groups

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Also, a statistically significant (P < 0.01) decrease in the EMG latency values was found in the injured nontreated group (1.003 ± 0.031) compared with the normal group (0.797 ± 0.002), and a statistically significant increase in the EMG latency values (P < 0.01) in the MSC-treated group (0.8127 ± 0.00235) and SC-treated group (0.8113 ± 0.00076) compared with the injured nontreated group. There were no statistically significant differences (P > 0.05) in the MSC-treated and SC-treated groups [Figure 2].


  Discussion Top


Peripheral nerve injuries are an economic burden for society in general and despite advanced microsurgical reconstruction of the damaged nerves, the functional result is unsatisfactory, with poor sensory recovery and reduced motor functions [15].

To analyze target organ reinnervation and to evaluate the conductivity of regenerated tissue, evoked muscle action potentials (MAPs) and the NCV of regenerated nerve were recorded. Evoked MAPs were calculated by recording the latency and the amplitudes of the MAP following stimulation of the regenerated nerves with supermaximal intensity.

After 8 weeks of standardized crush injury, the nontreated group show a significant decrease in the mean values of the NCV compared with the corresponding mean values in the normal control group; these results were in agreement with those of Büyükakilli et al. [16], who found that conduction velocity of the sciatic nerve in the crush group was significantly slower than that of the controls, but had not yet reached normal.

There was a significant decrease in the mean values of the amplitudes of the MAP compared with the corresponding mean values in the normal control group and an increase in the mean values of latencies compared with the corresponding mean values in the normal control group. This result was in agreement with that of Kiernan [17], who concluded that the amplitudes of the compound muscle action potientials (CMPs) following stimulation with supermaximal stimulation intensities of the regenerated nerves were significantly smaller than the maximal CMP amplitudes of the normal control group and latencies of the CMPs were larger at the regenerated sides compared with the normal control group.

After 8 weeks of standardized crush injury, the BMSCs-treated group showed a significant increase in the mean values of the NCV compared with the corresponding mean values in the injured nontreated group and an insignificant increase compared with the normal group.

Also, there was a significant increase in the mean values of the amplitudes of the MAP and a significant decrease in latencies in the BMSCs-treated group compared with the corresponding mean values in the injured nontreated group and an insignificant increase compared with the corresponding mean values of the normal group.

These results were in agreement with those of Takemura et al. [18]; in the BMSCs-treated group after crushed sciatic nerve injury, the NCV, amplitudes of the MAP, and latencies were similar to those of the normal control group because of their ability to secrete trophic factors such as BDNF. Zarbakhsh et al. [19] concluded that there were improvements in electrophysiological tests in the amplitudes and latencies of the BMSCs-treated group compared with the nontreated group of sciatic nerve injury as BMSCs can promote axonal regeneration in peripheral nervous system (PNS); these cells secrete many factors such as neurotrophic factors that induce tissue plasticity, such as NGF, BDNF, GDNF, CNTF, and VEGF, and also produce extracellular matrix proteins such as collagen I, collagen IV, fibronectin, and laminin. There is good evidence to support the hypothesis that transplantation of BMSCs may repair peripheral nerve injuries.

After 8 weeks of standardized crush injury, the SCs-treated group showed a significant increase in the mean values of the NCV compared with the corresponding mean values in the injured nontreated group and an insignificant increase compared with the normal group.

This results were in agreement with those of Mimura et al. [20], who concluded that the increase in the mean values of the NCV after SCs transplantation compared with the corresponding mean values in the nontreated group were caused by SCs, which can promote regeneration and remyelination of damaged nerve axons.

There was a significant increase in the mean values of the amplitudes of the MAP and decrease latencies in the SCs-treated group compared with the corresponding mean values in the injured nontreated group.

These results were in agreement with those of Bakhtyari et al. [21], who proved that there was a significant increase in the mean values of the amplitudes of the MAP and a significant decrease in latencies compared with the corresponding mean values in the injured nontreated group as SCs produce molecules such as laminin and collagen and express many cell adhesion molecules and receptors including neural cell adhesion molecule; they also synthesize neurotrophic molecules such as NGF, BDNF, and CNTF. These molecules and neurotrophic factors are essential for the survival of neurons and axonal regeneration. Krekoski et al. [22] reported that SCs are structural and functional cells that play a critical role in peripheral nerve regeneration; when the peripheral nerve is damaged, SCs synthesize and secrete a variety of neurotrophic factors and extracellular matrix that promote axonal growth.


  Conclusion Top


Transplantation of SCs and MSCs has some therapeutic effects

  1. Electrophysiological recordings improved compared with the corresponding mean values in the sciatic nerve injury nontreated group.
  2. It is a promising therapy for peripheral nerve injury and can be considered a lamp in the dark tunnel of peripheral nervous system injury.



  Acknowledgements Top


Conflicts of interest

None declared.

 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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