Bone Marrow Mesenchymal Stem Cells and Electroacupuncture Downregulate the Inhibitor Molecules and Promote the Axonal Regeneration in the Transected Spinal Cord of Rats

Abstract

Our previous study has reported that electroacupuncture (EA) promotes survival, differentiation of bone marrow mesenchymal stem cells (MSCs), and functional improvement in spinal cord-transected rats. In this study, we further investigated the structural bases of this functional improvement and the potential mechanisms of axonal regeneration in injured spinal cord after MSCs and EA treatment. Five experimental groups, 1) sham control (Sham-control); 2) operated control (Op-control); 3) electroacupuncture treatment (EA); 4) MSCs transplantation (MSCs), and 5) MSCs transplantation combined with electroacupuncture (MSCs + EA), were designed for this study. Western blots and immunohistochemical staining were used to assess the fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs) proteins expression. Basso, Beattie, Bresnahan (BBB) locomotion test, cortical motor evoked potentials (MEPs), and anterograde and retrograde tracing were utilized to assess cortical-spinal neuronal projection regeneration and functional recovery. In the MSCs + EA group, increased labeling descending corticospinal tract (CST) projections into the lesion site showed significantly improved BBB scales and enhanced motor evoked potentials after 10 weeks of MSCs transplant and EA treatment. The structural and functional recovery after MSCs + EA treatment may be due to downregulated GFAP and CSPGs protein expression, which prevented axonal degeneration as well as improved axonal regeneration.

Keywords

Spinal cord transection Bone marrow mesenchymal stem cells Electroacupuncture Transplantation Axonal regeneration

Introduction

Spinal cord injury (SCI) is a devastating clinical condition, and current treatments for SCI are of limited therapeutic effort because of the poor capacity of axonal regeneratation in the mammalian central nervous system (CNS). The failure of axonal regeneration following SCI has been attributed to nonpermissive environment containing inflammatory mediators, lack of neurotrophic support, inhibitory molecules, and formation of glial scar. Over the past two decades, numerous therapeutic strategies attempted to reduce these negative factors and promote axon regeneration, including transplanting tissue bridges (8,31), artificial scaffolds (48,61), cellular transplants of various cells types (3,5,26,27,29,40,45), growth factors (30,69), and even more various combinatorial approaches (19,28,38,39,49,55). Although some improvement has been achieved, an effective treatment of SCI is still lacking.

Bone marrow mesenchymal stem cells (MSCs) have been suggested as a promising candidate for the treatment of CNS injuries (13,46). Potential advantages of MSCs over other types of transplanted cells include their ability to be harvested from autologous donors, their relatively rapid expansion in vitro, and their possible availability for clinical use (13). MSCs also secrete a variety of growth factors and cytokines that could help reparation after CNS injury (6,51,59). Studies have suggested that MSCs can promote axonal regeneration and functional recovery after spinal cord injury attributed to MSCs secreting a variety of growth factors and providing directional guidance for regenerating axons (9,26). In addition, MSCs also can express different adhesion molecules (e.g., N-cadherin, laminin, fiberonectin) that may promote neuritogenesis in vitro (10,33).

Electroacupuncture (EA) is a therapeutic approach in which a needle inserted into an acupoint is attached to a trace pulse current that produces synthetic electric and needling stimulation. The application of EA for the treatment of SCI has shown promising results in the alleviation of patient suffering (65). EA is used on “Governor Vessel” acupoints for the treatment of SCI because the impairment of this channel is regarded as the essence of spinal cord damage in traditional Chinese medicine. The use of EA on the Governor Vessel has been shown to alleviate secondary damage after SCI in both patients and animal models (24,66). Interestingly, our previous study found that utilizing EA on the Governor Vessel promotes NT-3 secretion in the injured spinal cord and enhances the differentiation of grafted neural stem cells (NSCs) into neuron-like cells at the injured site (7).

Recently, our study reported that EA treatment could promote grafted MSCs survival and differentiation into neuron phenotype cells, and MSCs transplantation combined with EA treatment could promote 5-HT-positive and CGRP-positive axonal regeneration and partial hindlimb locomotor functional recovery in the spinal cord-transected rat (14). Recent studies also have reported that EA can reduce glial fibrillary acidic protein (GFAP) levels in the injured cord (15,50,68), which serves to inhibit reactive astrocyte proliferation and reduce glial scar formation. However, whether MSCs combined with EA treatment can promote axonal regeneration by regulating GFAP and extracellular matrix molecule (e.g., CSPGs and laminin) expression in the lesion site is not known.

In this study, we investigated whether MSCs transplantation could promote CST axonal regeneration and whether EA treatment could enhance this process through downregulating GFAP and inhibitory molecule CSPGs expression and upregulating promoting molecule laminin expression in the transected spinal cord, promoting structural, and functional recovery.

Materials and Methods

Preparation of Bone Marrow MSCs

The method for preparing MSCs has been described in our previous study (14). Briefly, the tibias and femurs of 1-month-old SD rats were dissected under anesthesia and aseptic conditions. After removing the end of the bone, 5 ml of Dulbecco's modified Eagle medium (DMEM, Gibco/BRL, Carlsbad, CA) was injected into the central canal of the bone to extract the bone marrow. The solution containing bone marrow was then centrifuged at 1000 rpm for 5 min. The pellet was resuspended in DMEM and supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml). The cells were then cultured in a 75-ml cell flask and, after plating the cells for 48 h, the medium was replaced to remove nonadherent cells. When the adherent MSCs grew near confluency, they were serially passaged using 0.25% trypsin/0.02% EDTA. After being passaged 3–5 times, the MSCs were ready for use in transplantation.

Animal Groups and Spinal Cord Surgery

A total of 85 adult female SD rats (230–250 g) (Experimental Animal Center, Zhongshan School of Medicine of Sun Yat-sen University, China) were used for this study. Rats were housed in a temperature (24 ± 2°C) and light-controlled (12:12 light/dark cycle) room with free access to food and water. Prior to experimental manipulation, rats were allowed to acclimate to the housing facilities and were handled daily at least for 3 days. All experimental protocols and animal handling procedures were approved by the Animal Care and Use Committee of Sun Yat-sen University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The rats were divided into five groups. The Sham-control group only received a laminectomy without spinal cord transection. The remaining four groups underwent complete transections at the T10 spinal segment of the spinal cord. The operated control (Op-control) group received a spinal cord transection only without any treatments; the EA group received EA treatment after the spinal cord transection; the MSCs group received MSCs transplantation after spinal cord transection; and the MSCs + EA group received transplanted MSCs plus EA treatment after transection.

For the T9–T10 laminectomy, the animals were anesthetized with 1% sodium pentobarbital (40 mg/kg, IP). The laminectomy was carried out at the T9–T10 level to expose the T10 spinal segment. The dura was cut and the T10 segment was transected completely with a scalpel with no tissue removed (14,64). The transection site was carefully examined under microscopy to ensure complete transection of the spinal cord. In the Op-control and EA groups, a piece of gelfoam (2 × 2 × 2 mm³, preinjected DMEM 5 μl) was inserted into the lesion site. In the MSCs and MSCs + EA groups, a small piece of gelfoam, which was preinjected with a MSCs suspension (1 × 10⁵ cells/μl, 5 μl), was placed into the transected site of the spinal cord. After surgery and gelfoam transplantion, the dura was sutured, and the muscle and skin were closed in layers. Rats in the Sham-control group received a laminectomy without any further spinal cord damage. After surgery, all rats received an intramuscular injection of penicillin (160,000 U/ml/day) and were housed separately in individual cages with enriched soft bedding. Manual emptying of the bladders was performed twice daily. In the EA and MSCs + EA groups, rats received EA treatment every other day, beginning the 7th day post-spinal cord transection for 7 weeks.

Electroacupuncture Treatment

Based on our previous work (14), two pairs of Governor Vessel acupoints were selected during EA treatment. One pair of acupoint is Changqiang (GV1) and Yaoshu (GV2); another pair of acupoints is Jizhong (GV6) and Zhiyang (GV9). The acupoint GV1 is located at the midpoint between the tip of the coccyx and the anus in prone position. GV2 is located on the posterior midline and in the hiatus of the sacrum in prone position. GV6 is located on the posterior midline and in the depression below the spinous process of the eleventh thoracic vertebra in prone position. GV9 is located on the posterior midline and in the depression below the spinous process of the seventh thoracic vertebra in prone position. GV6 and GV9 are located on the depression below the rostral and caudal spinous process of transected spinal cord, respectively. EA on GV6 and GV9 can directly treat the injured spinal cord. EA on GV1 and GV2 can improve emptying the bowels and bladder and reduce lumbar vertebrae pain. During EA treatment, rats were loosely immobilized in a restrainer specifically made for this treatment by exposing their dorsal spine, hind legs, and tails. Two pairs of stainless silver needles of 0.3 mm in diameter were inserted into the GV1 and GV2, and GV6 and GV9, to a depth of 5 mm. The two pairs of needles were then connected to the output terminals of an EA apparatus (Model G 6805-2, Shanghai Medical Electronic Apparatus Company, China). Alternating strings of dense-sparse frequencies (60 Hz for 1.05 s and 2 Hz for 2.85 s) were used. The intensity was adjusted to induce a slight twitch of the hindlimb (≤1 mA) and lasted for 20 min. EA was administered once every other day for 7 weeks, starting from the seventh day postsurgery.

Behavioral Analysis

The hindlimb locomotor behavior was evaluated by Basso, Beattie and Bresnahan's (BBB) open field locomotion test (2). The open field locomotor activity score was determined by observing and scoring behaviors involving the trunk, tail, and hindlimbs. Scores from two examiners were averaged. Scores ranged from 0 to 21 (0, no movement; 21, normal movement) (2). All animals (total 85; n = 18 in each group except Sham-control group at n = 13) received behavioral testing every 2 weeks postsurgery for 8 weeks. All behavioral tests were videotaped and both examiners were blind to each group when they participated in behavioral evaluation.

Anterograde and Retrograde Tracing

For anterograde tracing to assess the regeneration of corticospinal tract (CST), biotinylated dextran amine (BDA) was used (23,70). Eight weeks after the spinal cord surgery, 19 rats (n = 4 in each group except Sham-control at n = 3) were anesthetized by ketamine (40 mg/kg) and 1% sodium pentobarbital (30 mg/kg). A burr hole was drilled through the cranium to expose the sensorimotor cortex (SMC, 1–3 mm posterior to bregma and 1–3 mm lateral to bregma). A 10% solution of BDA (10,000 mw; Molecular Probes) was injected into eight sites at each SMC (0.5 μl/site) covering the hindlimb region. Two weeks after BDA injection, the rats were anesthetized and perfused with 4% paraformaldehyde in 0.1 M PB (pH 7.4). The T8–T12 segments of spinal cords were collected and sliced in the horizontal plane at 25 μm thickness on a freezing microtome. The selected sections were incubated with Cy3–conjugated streptavidin to visualize BDA-containing corticospinal axons. The sections were examined under the fluorescence microscope.

For retrograde tracing, fluorogold (FG, 3%) was used to label neurons in the sensorimotor cortex (SMC) and red nuclei (RN) (70). Nine weeks after the spinal cord surgery, the spinal cords of 19 rats (n = 4 in each group except the Sham-control group at n = 3) were exposed at 5 mm caudal (about T12 spinal segment) from the lesion site and 3% FG was bilateral injected into two sites (0.5 mm lateral from the midline of spinal cord, 0.3 μl/site) using stereotaxic apparatus. One week later, the rats were perfused by 4% paraformaldehyde in 0.1 M PB. The brains were cut into 25-μm-thick serial sections in the coronal plane and collected 5 sets of sections by every 5th section mounted. One set of sections was examined using a fluorescence microscope. The FG positively labeled neuron was characterized by the bright signal in the cell body and process in comparison with the background environment with morphological features of a neuron.

Quantitative analysis of NF- and BDA-positive nerve fibers was performed based on our previous study (14). Briefly, a calibrated reticle eyepiece was used to delineate regions 300 μm rostral to the transection site, directly at the transection site, and 300 μm caudal to the transection site [see the supplementary additional file 1 in (14), available at the BMC Neuroscience or PubMed website to clarify the location of quantification]. The positive nerve fiber profiles were quantified in all regions at 200× magnification. Ten sections per rat were analyzed and the total number of labeled fibers in all regions of each group was averaged.

Quantitative analysis of FG-labeled neurons was performed in SMC and red nuclei (RN). The number of FG-labeled inner pyramidal cells was counted in 2 unit areas (0.16 mm² each) in the left and right SMC. Twenty sections from the cortex and 20 sections from the red nucleus per rat were analyzed. Autofluorescence was distinguished from true tracer labeling by examining sections with all three filters. The neurons that only FG positive under the UV-2A filter were counted.

Electrophysiology

Eight weeks after the spinal cord surgery, 27 rats (n = 6 in each group except the Sham-control at n = 3) were deeply anesthetized with ketamine (40 mg/kg) and 1% sodium pentobarbital (30 mg/kg). Motor-evoked potentials (MEPs) were elicited by electrical stimulation of the SMC located 2 mm lateral to midline and 2 mm caudal to bregma (62). A single pulse stimulation 50 ms in duration, 5.1 Hz in frequency, and 0.01 V in voltage. MEPs were recorded simultaneously at the T12 and L1 levels of the spinal cord using bipolar electrodes (active silver midline epidural electrodes; reference needle electrodes inserted into the paraspinal muscles). At least 2 MEP averages of 50 responses were obtained and stored (BL-410E Data Acquisition Analysis System for Life Science, Taimeng, China) for each animal with stimulation on both sides of SMC (gain parameter 2000, time constant 0.01 s, filtering 300 Hz). The presence or absence of MEPs at each level was confirmed and the amplitude and latency of recorded averages were analyzed.

To evaluate the degree of regenerating descending nerve fiber control of hindlimb locomotor function, 5 rats (one rat from each group) were used to perform spinal cord retransection after recording MEPs. The animal was incised to expose the original transection site (between T9 and T10). The whole graft tissue was removed using a microscissor. MEPs tests were performed again 2 weeks later. After all of the performances, the animals were perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4) for immunohistochemical staining.

Immunohistochemical Staining

The selected sections were rinsed with PBS, then the sections were blocked with 10% normal goat serum (NGS) with 0.3% Triton X-100 in 0.01 M phosphate-buffered saline (PBS, pH 7.4) for 30 min at 37°C and incubated overnight at 4°C with primary antibodies. The omission of primary antibodies served as the negative control. Primary antibodies were used with the following: mouse anti-neurofiliment (NF200, 1:300, Boster, China), mouse anti-chondroitin sulfate clone CS-56 (CSPG; 1:400, Sigma), rabbit polyclonal anti-laminin (1:300, Boster, China), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:80, Sigma). Subsequently, the sections were rinsed with PBS and incubated with their respective secondary antibodies for 1 h at 37°C. Cy3-conjugated goat anti-mouse IgG and Cy3-conjugated goat anti-rabbit IgG (1:800, Jackson Immunoresearch Labs, Inc.), FITC-conjugated goat anti-mouse IgG, and FITC-conjugated goat anti-rabbit IgG (1:200, Jackson Immunoresearch Labs, Inc.) were used as secondary antibodies, respectively. Finally, the sections were rinsed with PBS and coverslipped. The stained sections were examined with a Leica fluorescence microscope and images were captured.

Nissl Staining and Neuron Counting

One set of brain sections taken from 19 animals for FG retrograde labeling groups and the cross sections of L1 spinal cord segment taken from 19 rats for BDA antrograde labeling groups were stained with neutral red (i.e., Nissl staining). The neuronal densities of the inner pyramidal layer of SMC and RN were counted under grid-equipped microscopy based on stereological principle. Neurons of Clarke's nuclei (CN) on 50 serial spinal cord sections of each rat were also counted.

Western Blot Analysis

Western blot analysis was performed at 8 weeks after surgery. The spinal cords of 20 rats (4 rats/group) were immediately removed, and the spinal segments (0.5 cm) containing the lesion site were dissected and homogenized on ice in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Biolytes pH 5–8, 0.5% Triton X-100, and 1% cocktail) using homogenizers and a sonication probe. Homogenates were centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was collected and stored at −80°C for Western blotting. The protein concentration was assayed according to the Bradford method with BSA as the protein standard. Equal amounts of protein (50 μg) were then loaded on a 10% SDS polyacrylamide gel (SDS-PAGE) followed by electrophoresis for 1 h at 150 V. Proteins were transferred from the gel to a nitrocellulose membrane for 2 h at 250 mA. After blocking nonspecific binding sites with 5% nonfat milk in TBST (0.5% Tween 20 in TBS) for 1 h at room temperature, the membranes were incubated with rabbit anti-GFAP (1:1500, Sigma), mouse anti-CSPGs (1:1000, Sigma), rabbit anti-laminin (1:500, Boster), and rabbit anti-β-actin (1:1000, cell signaling, USA) overnight at 4°C, respectively. After washing, the members were treated with a horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit or goat anti-mouse, 1:5000, Jackson, USA) for 2 h at room temperature, and then washed thoroughly. The protein bands were visualized by enhanced chemiluminescence (ECL) detection reagents (Applygen Technologies Inc., Beijing, China) and exposed onto X-films for 1–5 min. The areas and densities of protein bands were quantified using Scion image software (version 4.0). The value of band densities for samples was then expressed by the ratio of relative intensity to the level of β-actin.

Data Analysis

Data are given as mean ± SD. Statistical analyses were performed using one-way ANOVA or repeated measure ANOVA. If equal variances were found, Fisher's least significant difference test was performed. Otherwise, the Dunnett's T3 was used. Multiple linear regression was used to analyze the correlation of locomotor function with the density of FG-labeled neurons. Differences were considered statistically significant at p < 0.05.

Results

Behavioral Assessment

After spinal cord transection, all rats were paralyzed and moved by pulling their forelimbs. They also displayed urinary and fecal incontinence. BBB open field test indicated the hindlimb locomotor activity improved gradually in the three treatment groups throughout the entire follow-up survival period by showing BBB scores gradually increasing over time in each group. Statistic analysis revealed that BBB scores were significantly higher in the three treatment groups compared to the Op-control group from 2 to 8 weeks postlesion (p < 0.05), and the MSCs + EA group showed the highest score at the 8th week (p < 0.01) (Fig. 1).

Figure 1.

BBB scores were obtained starting from the first week throughout the 8-week survival period. The scores gradually increased with time in each group. BBB scores were significantly higher in the three treatment groups compared to that of the Op-control group from 2 to 8 weeks posttransection (p < 0.05). The MSCs + EA group showed a significantly higher score than the other treatment groups beginning the 4th week following transection. Especially at 8 weeks after the operation, the scores of the MSCs + EA group were significantly the highest (^*p < 0.01 compared to the Op-control group; #p < 0.01 compared to the EA group/MSCs group). Data are mean ± SD.

Motor Evoked Potentials (MEPs)

Representative sample traces showed that MEPs stimulation evoked a short-latency positive-negative-positive wave, which was absent in the Op-control group and was partially restored by EA, MSCs, or MSCs + EA combined treatment at the end of the 8th week following transection (Fig. 2A). The restored MEPs were abolished by retransection (Fig. 2B), indicating descending axonal regeneration. The mean number of the latency and amplitude are presented in Table 1. The latency of MEPs was significantly prolonged in all spinal cord transection groups compared with the Sham-control group (p < 0.05). Although MEPs in the MSCs + EA group displayed shorter latency, there was no significant difference compared to the MSCs or EA group. However, the amplitude of MEPs was significantly higher in the MSCs + EA group than that in the MSCs or EA group (p < 0.05) (Table 1).

Figure 2.

Motor evoked potentials (MEPs) recovery after MSC transplantation and EA treatment at the end of 8 weeks after transection of spinal cord. (A) The MEPs of the rats in the Op-control group were absent. After receiving EA, MSCs, or the MSCs + EA treatment. (B) The restored MEPs were abolished by retransection.

Table 1.

Comparisons of the Latency and Amplitude (Mean ± SD) of MEPs Among All Groups

Groups	No.	Latency (ms)	Amplitude (mV)
Sham-control group	3	14.69 ± 1.03	0.29 ± 0.03
Op-control group	6	0.00 ± 0.00	0.00 ± 0.00
EA group	6	26.30 ± 2.30^*	0.14 ± 0.02^*
MSCs group	6	27.48 ± 3.77^*	0.12 ± 0.02^*
MSCs + EA group	6	24.43 ± 2.69^*	0.19 ± 0.02^{* †}

EA, MSCs, or MSCs + EA groups versus Sham-control group, p < 0.05 by one-way ANOVA.

†

MSCs + EA group versus MSCs or EA groups, p < 0.05 by one-way ANOVA.

Axonal Regeneration

Axonal regeneration was assessed by anterograde/retrograde labeling and neurofilament immunohistochemistry. Neurofilament-200 (NF) immunostaining showed that there were few NF-stained fibers in the tissue surrounding the cysts in the vicinity of the lesion site in the Op-control group (Fig. 3A, a). Some NF-positive fibers appeared in the lesion site in the EA or MSCs group (Fig. 3B, b, C, c) and a significant more number of NF-positive fibers appeared in the MSCs + EA group (Fig. 3D, d). Quantification showed NF-positive fibers located in the rostral region nearby the lesion site, the lesion site itself, and the caudal region nearby the lesion site (Table 2). Statistical analysis indicated that the MSCs + EA group has a significantly higher number of NF-positive fibers located in the three regions compared to that of the EA or MSCs group (p < 0.01). However, the number of NF-positive fibers was not significantly different between the MSCs group and EA group (p > 0.05) (Table 2).

Figure 3.

The expression of Neurofilament-200 (NF) by immunohistochemistry staining. (A–D) A horizontal section of spinal cord shows NF-positive fibers in the Op-control group (A), EA group (B), MSCs group (C), and MSCs + EA group (D). (a–d) Higher magnification of the rectangle boxes in (A–D). In the Op-control group, few NF-positive fibers (arrow) appeared in the lesion site, with some cysts in the vicinity. In the EA and MSCs groups, some NF-positive fibers (arrows) showed in the lesion site. In the MSCs + EA group, more NF-positive fibers (arrows) showed in the central lesion site. Scale bars: 320 μm (A–D); 40 μm (a–d).

Table 2.

Comparison of the Number of NF-Positive Fibers (Mean ± SD) in the Lesion Site and the Region Immediately Rostral/Caudal to the Lesion Site Among the Four Groups

Groups	No.	Rostral Region	Lesion Site	Caudal Region
Op-control group	5	8.06 ± 2.82	0.34 ± 0.36	6.42 ± 2.30
EA group	5	19.96 ± 5.99^*	5.46 ± 1.65^*	15.42 ± 5.26^*
MSCs group	5	18.82 ± 3.34^*	5.32 ± 1.07^*	16.64 ± 2.99^*
MSCs + EA group	5	30.96 ± 4.40^{* †}	14.20 ± 6.88^{* †}	22.54 ± 3.96^{* †}

^*EA, MSCs, or MSCs+EA groups versus Op-control group, p < 0.01 by one-way ANOVA.

^†MSCs + EA versus MSCs or EA groups, p < 0.01 by one-way ANOVA.

Axonal regeneration in the corticospinal tract (CST) was assessed by injecting BDA into the sensorimotor cortex. In the Op-control group, cystic cavities appeared in the vicinity of the lesion site and no BDA-labeled axons reached to the rostral host/lesion interface (Fig. 4A, A1). In the MSCs group and the EA group, some BDA-positive axons were found in the proximal stump of the spinal cord and reached the rostral host/lesion interface, but no BDA-positive axons crossed the rostral host/lesion interface into the lesion site (Fig. 4B, B1, C, C1). However, in the MSCs + EA group, more BDA-positive axons were shown crossing the rostral host/lesion interface and extending into the grafts site but did not pass through the caudal lesion/host interface (Fig. 4D, D2). Moreover, these CST axons had regenerating branches and some of them projected sprouts into the gray matter (Fig. 4D1). As expected, in the Sham-control group the prominent BDA-positive axons were parallel with the longitudial axis of spinal cord and arranged in a tight bundle (Fig. 4E). The quantification of the BDA-positive axons in the region 0.3 mm rostral to the lesion site indicates the number of the BDA-positive axon was dramatically increased in the MSCs + EA (7.00 ± 2.48) group compared to the Op-control group (0.75 ± 0.95), EA group (5.25 ± 1.50), or MSCs group (2.87 ± 0.63) (p < 0.01) (Fig. 4F). But only two of the four MSCs + EA animals had visible BDA-positive fibers in the middle of lesion site (2.43 ± 2.41).

Figure 4.

The regeneration of BDA anterogradely labeled corticospinal tract (CST). (A-D) A horizontal section of spinal cord shows BDA-labeled CST in the Op-control group (A), EA group (B), MSCs group (C), and MSCs + EA group (D). Asterisks (^*) mark the transection sites, which are outlined by dotted lines. In the Op-control group, BDA-labeled CST was distantly away from the host/lesion interface rostral to the lesion site (A), and few BDA-labeled CST were found in the rostral region to the lesion site (A1). In the MSCs group (C) and the EA group (B), some labeled CST fibers (arrows) were found in the proximal stump of the spinal cord (B1 and C1), but no BDA-labeled fiber crossed the host/lesion interface into the lesion site. In the vicinity of the lesion, these fibers displayed typically tortuous (D, D1) and easily distinct from the normal straight fibers in the rostral CST (E). In the MSCs + EA group, BDA-labeled CST axons (arrows) reached the grafts (D2). D1, D2 is the higher magnification of rectangle boxes (1 and 2) in (D). Scale bars: 320 μm (A-D), 40 μm (A1–D2, E). (F) Comparison of the number of BDA-labeled CST in the lesion site and the region immediately rostral to the lesion site among the four groups (EA or MSCs + EA groups vs. Op-control group, ^*p < 0.01; MSCs + EA group vs. MSCs group, #p < 0.05). Data are mean ± SD.

Axonal regeneration was also assessed by retrograding transport of FG from an injection site of the caudal cord. In the Sham-control group, many FG-labeled neurons were present in bilateral SMC (Fig. 5A) and RN (Fig. 5a) that project to the spinal cord, and the shape of FG-labeled neurons was very clear. However, there were few FG-labeled neurons in SMC (Fig. 5B) and RN (Fig. 5b) in the Op-control group. In the MSCs group and EA group, some FG-labeled neurons were found within the SMC (Fig. 5C, D) and RN (Fig. 5c, d). In the MSCs + EA group, more FG-labeled neurons in the SMC and RN were found compared to other groups (Fig. 5E, e). The quantification of the FG-labeled neurons in the SMC indicated the number of the FG-labeled neurons in unit area were dramatically increased in the MSCs + EA (4.90 ± 0.82) group compared to the Op-control group (0.00 ± 0.00), EA group (2.50 ± 0.91), or MSCs group (2.13 ± 0.48) (p < 0.05) (Fig. 5F). The number of the FG-labeled neurons in RN were also dramatically increased in the MSCs + EA group (6.70 ± 2.31) compared to the Op-control group (0.13 ± 0.25), EA group (4.62 ± 1.80), or MSCs group (3.38 ± 0.48) (p < 0.05) (Fig. 5F). Moreover, the linear regression analysis showed that there was a positive correlation between the number of FG-labeled neurons in supraspinal nuclei (including SMC and RN) and the BBB scores of individual animals (p < 0.001) (Fig. 5G, H).

Figure 5.

Fluorogold (FG) retrogradely labeled neurons in the sensorimotor cortex (SMC) (A-E) and the red nucleus (RN) (a-e). In the Sham-control group, the shape of FG-labeled neurons in the SMC (A) and RN (a) was prominent, but no FG-labeled neurons were seen in the SMC or RN of the Op-control group (B, b). In the EA group and MSCs group, there were a few FG-labeled neurons (arrows) within SMC (C, D) and RN (c, d). However, in the MSCs + EA group, the significant numbers of FG-labeled neurons (arrows) were identified within SMC (E) and RN (e). Scale bars: 20 μm. (F) Comparison of the number of FG-labeled neurons in SMC and RN among the four groups (EA, MSCs, or MSCs + EA vs. Op-control group, *p < 0.05; MSCs + EA vs. MSCs or EA groups, #p < 0.05). (G) Correlation between the number of the FG-labeled neurons in SMC and hindlimbs locomotor function (BBB scores) of the rat. Linear regression analysis showed positive correlations between the FG-labeled neurons in SMC and BBB scores (correlation coefficient is 0.95, p < 0.001). (H) Linear regression analysis showed positive correlations between the FG-labeled neurons in RN and BBB scores (correlation coefficient is 0.88, p < 0.001). Data are mean ± SD.

Neuronal Survival

To evaluate the neuronal degeneration and survival after spinal cord injury, we selected SMC, RN, and CN of the L1 spinal segment to represent the pyramidal system and the extrapyramidal system, respectively. Cell counting showed significantly more surviving neurons in the SMC, RN, and the CN in the tree treatment groups compared to those in the Op-control group. In particular, the MSCs + EA group preserved more surviving neurons than the other groups (p < 0.05) (Fig. 6).

Figure 6.

Neutral red staining (i.e., Nissl staining) was performed to assess the surviving neurons in the sensorimotor cortex (SMC), the red nucleus (RN), and Clark's nuclei (CN). Scale bars: 40 μm. Cell counting showed that the numbers of surviving neurons were significantly higher in the SMC, RN, and CN in the tree treatment groups compared to the Op-control group (*p < 0.05). Specifically, the MSCs + EA group preserved the highest number of surviving neurons (EA, MSCs, or MSCs + EA groups vs. Op-control group, *p < 0.05; MSCs + EA group vs. MSCs group, #p < 0.05). Data are mean ± SD.

GFAP Expression

GFAP immunohistochemistry showed that GFAP-positive cells were located mainly in the host tissue surrounding the injury and cystic cavities; few GFAP-positive cells were located in the central lesion site in all spinal cord transection groups. To examine the interaction of axons regeneration and glial scar, immunofluorescence double-labeling for NF/GFAP (Fig. 7A1–B3) and BDA/GFAP was performed on the MSCs + EA group. NF-positive/BDA-labled CST axons partially extended across the GFAP-rich host/lesion interface and penetrated cell grafts (Fig. 7C1–C3).

Figure 7.

NF/GFAP (A1–A3) and BDA/GFAP (C1–C3) immunofluorescence double-labeling revealed the interaction of axonal regrowth and glial scar in the MSCs + EA group. (B1–B3) Higher magnification of the rectangle boxes in (A1–A3). NF-positive/BDA-labled CST axons (arrows) partially extended across the GFAP (green)-rich host/lesion interface and penetrated cell grafts (A3, B3, C3). Scale bars: 20 μm. (D, E) The level of GFAP protein expression in the injured spinal cord by Western blot. The level of GFAP protein was quantified by chemiluminescence and normalized by the loading control (β-actin). The levels of GFAP protein in the EA group and MSCs + EA group were significantly downregulated (Op-control group vs. Sham-control group, Δp 0.05; EA or MSCs + EA groups vs. Op-control group, ^*p < 0.05; MSCs + EA group vs. MSCs group, #p < 0.05). Data are mean ± SD.

Western blot analysis showed that the levels GFAP protein was higher in the Op-control group compared to the other groups (p < 0.05). The levels of GFAP protein were significantly lower in the EA group and MSCs + EA group (Fig. 7D, E), suggesting that EA or EA combining with MSCs treatment decreased astrocyte proliferation after injury.

CSPGs and Laminin

We found that CSPGs were mainly expressed in both the host tissue surrounding the lesion site and the lesion site itself (Fig. 8). Immunolabeling CSPGs in those regions was significantly increased in the Op-control group (Fig. 8A2) compared to the other three groups (Fig. 8B2, C2, D2). Notably, the CSPGs expression was the least in the MSCs + EA group (Fig. 8D2). The axonal regeneration promoting factor laminin immunolabeling was also observed in both the host tissue surrounding the lesion and the lesion regions (Fig. 8). Some laminin colocalized with CSPGs within grafts (Fig. 8A3–D3). In the Op-control group, the laminin expression was less than other groups (Fig. 8A1). However, laminin expression was increased in both of the MSCs (Fig. 8C1) and MSCs + EA groups (Fig. 8D1).

Figure 8.

Immunofluorescence double-labeling was performed to assess the expression of laminin (green) and CSPGs (red) in the horizontal section of spinal cord. (A1–A3) Op-control group, (B1–B3) EA group, (C1–C3) MSCs group, and (D1–D3) MSCs + EA group. In the Op-control group, the laminin expression (A1) was less than other groups and CSPGs expression (A2) was more than other groups. The extent of CSPGs expression in the host spinal cord surrounding the lesion site was gradually attenuated in the EA (B2), MSCs (C2), and MSCs + EA (D2) groups, and the extent of laminin expression was increasing in the EA (B1), MSCs (C1), and MSCs + EA (D1) groups. Laminin structures within grafts also colocalized with CSPGs-labeled structures (A3, B3, C3, D3). Scale bar: 160 μm. (E, F) The levels of CSPGs and laminin protein expression in the injured spinal cord by Western blots. The levels of CSPGs and laminin proteins were quantified by chemiluminescence and normalized by the loading control (β-actin). Compared with the Op-control group, the levels of CSPGs protein in the EA group and MSCs + EA group were significantly downregulated. However, the levels of laminin protein showed no difference between groups (Op-control group vs. Sham-control group, Δp < 0.05; EA or MSCs + EA groups vs. Op-control group, ^*p < 0.05). Data are mean ± SD.

Western blot analysis showed that the levels of CSPGs protein were significantly lower in the EA group and MSCs + EA group compared to the Op-control group (p < 0.05) (Fig. 8E, F). However, the levels of laminin protein were not different between groups (p > 0.05) (Fig. 8E, F).

Discussion

In this study, we confirmed and extended our previous work by investigating the effect of MSCs transplantation with EA treatment on structural and functional recovery of the injured spinal cord. We found that BBB scales and motor evoked potentials were significantly improved and the number of NF-positive and BDA-labeled CST axons in and near the lesion site of the injured spinal cord were significantly increased in the MSCs + EA-treated group compared to other groups that underwent MSCs transplantation or EA treatment alone. Furthermore, CSPGs and GFAP protein expressions were decreased, correlating with neuroregeneration and functional recovery in the MSCs + EA group.

Our results showed that the significant improvement of BBB scales and MEPs and the significantly increasing number of NF-positive and BDA-labeled CST axons in and near the lesion site of the injured spinal cord in the MSCs + EA group were consistent with our previous study where we showed that increasing 5-HT and CGRP-positive axons in the transplant site improved hindlimb locomotion in the MSCs + EA group (14). The CST is one of the most important brain tracts that controls motor function in humans; its role is not well defined in rats yet. Several studies have reported that the regeneration of lesioned CST axons can be promoted by application of stem cells (60) and neurotrophic growth factors, such as BDNF (63) and NT-3 (22) in different acute spinal cord injury models. Accordingly, our previous studies have shown that the combined treatment of MSCs and EA increased the NT-3 level of the injured spinal cord (14). In addition to the increasing number of BDA anterograde labeled CST into the lesion site in the MSCs + EA treated group, there were more FG-labeled neurons present in bilateral SMC and RN. These data suggested that more descending axons regenerated across the lesion site and reached the caudal cord in the MSCs + EA group. The multiple linear regressions showed a positive correlation between the number of FG-labeled supraspinal nuclei (including SMC and RN) and BBB scores. These results are consistent with another study (25). The axonal regeneration after MSCs plus EA treatment may provide a structural basis for reestablishment of physiological signal transduction across the lesion site to the caudal cord.

Spinal cord transection injures neurons, but if the bodies of axotomized neurons survive, their axons could have the ability to sprout or regrow. Therefore, the survived number of axotomized neurons in the SMC, RN, and CN can indirectly reflect the axonal regeneration ability. In the present study, we found there were more neuron numbers in the SMC, RN, and CN in the MSCs + EA group. The results suggested that MSCs + EA combined treatment could promote neuronal survival.

Another important finding in the present study is the decreased GFAP and CSPGs expression in the MSCs + EA-treated group. It is known that the failure of axonal regeneration after SCI has been attributed in part to the nonpermissive environment of the glial scar (18). The glial scar is predominately formed by reactive astrocytes (18). Although the extracellular matrix (ECM) molecules produced by astrocytes have been shown to inhibit axonal regeneration (1,12,41), astrocytes also have been shown to secrete permissive ECM molecules that promote axonal regrowth (42). Thus, astrocytes may promote or inhibit regeneration after SCI depending upon the balance of growth-inhibiting and growth-promoting ECM molecules that they produce. CSPGs are probably the most important of the inhibitory molecules produced by reactive astrocytes (17,18,56). In vivo and in vitro studies have shown that neurons cease to extend their axons into areas rich in CSPGs (11,12,42,71). Mutant mice that are deficient in both GFAP and vimentin (important cytoskeletal proteins that are induced in reactive astrocytes) show reduced astroglial reactivity, increased supraspinal sprouting, and improved functional recovery after spinal cord hemisection (43,53). Intrathecal administration of chondroitinase ABC (ChABC, degrading CSPG glycosaminoglycan) following spinal cord injury promoted the regeneration of various axon tracts as well as some recovery of function (4,44). These studies indicate that downregulating GFAP and CSPGs expression can promote axonal regeneration after spinal cord injury. Therefore, our data suggested that downregulating of GFAP and CSPGs inhibitory molecules could be one of the many possibilities that promote the axonal regeneration after MSCs transplantation plus EA treatment.

The underlying mechanism of MSCs transplantation plus EA treatment reducing GFAP and CSPGs proteins expression is still not very clear. MSCs may secrete a certain nutrition that can inhibit GFAP and promote axonal sprouting (20,34,54,67). Studies that indicate MSCs modifying ischemia-induced astrocytic activation and reduced GFAP expression in astrocytes in vitro (20), reducing the thickness of the scar wall (34), and reducing the expression of inhibitory glycoproteins, thereby creating a permissive environment for neurite outgrowth (54), support this idea. In addition, MSCs produce membrane type I matrix metalloproteinase (MT-1 MMP) and matrix metalloproteinase 2 (MMP2) (58). Both enzymes can degrade CSPGs (such as aggrecan and versican) (16,47). A recent study has reported that MSCs transplantation can increase the activity of tissue plasminogen activator (tPA) and decrease the expression of the tPA inhibitor plasminogen activator inhibitor (PAI-1) in astrocytes in the peri-infarct area of ischemic brain and further enhance axonal growth (67). The tPA degrades the extracellular matrix (ECM) directly by removing glycoproteins from the ECM (32). Thus, MSCs transplantation reduces the glial scar and the expression of inhibitory glycoproteins (CSPGs) by directly expressing matrix metalloproteinases to degrade inhibitory molecules or by increasing tPA in reactive astrocytes. On the other hand, studies have reported that EA can reduce the formation of glial scar and the expression of epidermal growth factor receptor (EGFR) and GFAP in the injured spinal cord, and promote axonal regeneration (50,68). In the adult CNS, the EGFR pathway is absent from astrocytes (21), but is highly upregulated and activated following injury (35,36,52). In response to activation of the EGFR pathway, astrocytes increase synthesis of GFAP (37) and CSPGs (57). Therefore, EA treatment can decrease the expression of GFAP and CSPGs by inhibiting the EGFR expression in reactive astrocytes. Thus, MSCs transplantation plus EA treatment may synergistically downregulate expression of inhibitory molecules GFAP and CSPGs by combined their capabilities, and synergistically modify the hostile environment in the lesion site to promote axonal regrowth.

In summary, the results from this study along with our previous study (14) indicate that MSCs transplantation combined with EA treatment not only increased MSCs survival and differentiation (14), but also promoted the CST regenerated into injured site and functional improvement, perhaps due to the increase of NT-3 and cAMP levels (14) and downregulation of inhibitory molecules GFAP and CSPGs. Our results suggest a new therapeutic potential of the combination of MSC transplantation with EA treatment to treat spinal cord injury patients.

Footnotes

Acknowledgments

This research was supported by a research grant from the Chinese National Natural Science Foundation (No. 30472132, 30973721) and the research grants of the Administrative Bureau of Chinese Traditional Medicine of Guangdong Province (No. 1050167; 2007109) to Y. S. Zeng.

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