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Implanted spike wave electric stimulation promotes survival of the bone marrow mesenchymal stem cells and functional recovery in the spinal cord injured rats
Neuroscience Letters 491 (2011) 73–78
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Implanted spike wave electric stimulation promotes survival of the bone marrow mesenchymal stem cells and functional recovery in the spinal cord injured rats Wenliang Wu a , Hua Zhao a , Bin Xie b , Haichun Liu a , Yunzhen Chen a,∗ , Guangjun Jiao a , Hongliang Wang a a b
Department of Trauma Surgery, Shandong University Qilu Hospital, Wenhuaxi Road 107, Jinan 250012, China Department of Spine Surgery, Weihai Hospital, China
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Article history: Received 29 October 2010 Received in revised form 27 December 2010 Accepted 4 January 2011 Keywords: Mesenchymal stem cells Electric field stimulation Spinal cord injury
a b s t r a c t Transplantation of bone marrow-derived mesenchymal stromal cells (BMSCs) into the injured spinal cord may provide therapeutic benefit, but its application is limited by their poor survival and low differentiation rate into neurons. Electrical stimulation (ES) has been reported to promote survival and differentiation of the BMSCs. Therefore we investigated whether implanted spike wave ES could improve survival of BMSCs after transplantation and result in functional improvement in animals with spinal cord injury. Our results showed that the number and ratio of survived BMSCs near the lesion site were significantly increased in the BMSCs + ES-treated group as compared to BMSCs transplantation or ES treatment alone group. Furthermore, results from BBB scales, SSEP and DTI demonstrated a significant improved functional recovery in the BMSCs + ES group. This indicated that implanted spike wave ES could promote the bioactivity of BMSCs and their survival. This represents a new therapeutic potential of the combination of BMSCs transplantation with implanted spike wave ES to treat spinal cord injury. © 2011 Elsevier Ireland Ltd. All rights reserved.
Severe spinal cord injury (SCI) usually results in long-lasting deficits, involving partial or complete paralysis and loss of sensation below injury level. These deficits result from neuronal damage, demyelination of axons and dysfunctional neruoglial cells, all of which the central nervous system (CNS) has a limited ability to repair itself [24]. The failure of axonal regeneration following SCI has also been attributed to a non-permissive environment containing inflammatory mediators, lack of neurotrophic support and formation of glial scars [10,12]. It has been suggested that transplantation of bone marrowderived mesenchymal stem cells (BMSCs) into the injured spinal cord may provide therapeutic benefit [24,5,6,8,19,20,30]. One possible mechanism is that BMSCs can differentiate into neural lineage cells under specific in vitro conditions [20,17,26,4]. However, these differentiated “neurons” are only identified to resemble true neurons phenotypically but different in electrophysiological characteristics [25,7]. Second, BMSCs secrete a broad spectrum of bioactive macromolecules that could be immunoregulatory and contributing to structure regenerative microenvironments in fields of injury [19,20,4]. Other possible mechanisms include remyelination and a bridging effect for axon regeneration [1]. Despite these
∗ Corresponding author. Tel.: +86 13606373827. E-mail address: [email protected] (Y. Chen). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.01.009
advantages, the application of BMSCs is limited by their poor survival and low differentiation rate into neurons when transplanted into injured spinal cord [20,7,2]. The use of electrical stimulation (ES) has been involved in restoring the lost functions following spinal cord injury for decades. Several therapeutic devices are commercially available at present, including devices for functional electrical stimulation (FES) [11], direct current stimulation, oscillating field stimulation (OFS) [21], and electro-acupuncture (EA) [8]. The common background for these therapies is the consensus that electrical fields (EFs) serve as important physiological signals and play a crucial role in the nervous system development and regeneration [11,14,23]. Numerous researches have also investigated the effect of ES on stem cells. Weak direct current (DC) has been confirmed to regulate stem/progenitor migration or differentiation [3,15]. Alternating current could also exert improved effects on neural stem cell viability and differentiation [16]. Furthermore, EA has been reported to promote survival and differentiation of the BMSCs in rats [8]. Thus, we hypothesize that maybe implanted electrodes releasing ES in vivo could have the same effects on BMSCs in vitro. Silver spike point therapy (SSP), which is also known as needlefree electro-acupuncture therapy, has been confirmed to regulate pain by generating a physiological spike wave with bi-directional symmetrical waveforms [13]. It is reasonable that the physiological low-frequency spike wave could also prolong BMSCs survival in
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Fig. 1. The survival of BMSCs in the injured spinal cord. BrdU-labeled (brown, red arrow) BMSCs were well integrated with host tissue. (a) BMSCs-treated group; (b) BMSCs + ES-treated group; (c) the number of survived BMSCs within 10 mm of the lesion site per unit area (**p < 0.01); (d) the ratio of survived BMSCs within 10 mm of the lesion site (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
injured spinal cord as EA did. To our knowledge, this combination use of BMSCs transplantation and implanted spike wave ES has not been investigated before. In this study, we first examined whether implanted spike wave ES could exert effects on BMSCs survival after transplantation in rats spinal cord injury model. Then we investigated whether this combined treatment resulted in any functional improvement than single treatment of each. Our results demonstrated that implanted physiological spike wave could prolong BMSCs survival and improve functional recovery after BMSCs transplantation. Following harvesting BM aspirates from the femur of adult male Sprague–Dawley rats (Animal Center of Shandong University Medical School, Jinan, China), mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep, Fresenius Kabi Norge, AS) and passaged as routine. For immunohistological tests, cells were incubated with 10 mol/L 5-bromo-2-deoxyuridine (BrdU, Sigma–Aldrich, USA) for 3 days before transplantation. The device for ES was designed using s a multi-frequency SSP machine (Felicia TM21, Nihon Medix, Japan). The electrodes for implantation were made of standard pacemaker cables and plantinum/iridium tips with a 2-mm2 surface area. The spike wave stimulation generated was the same as that used in traditional EA. All protocols were approved by Shandong University Committee on Animal Research. Animals were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP). A laminectomy was performed at the T10 spinal cord level with duramatter intact. Contused incomplete SCI was induced by dropping a 10-g weight rod from a 6 cm height onto the exposed dorsal surface of the spinal cord using an NYU impactor. One week after SCI, the injured rats were randomly assigned to four groups: BMSCs-treated (n = 15), ES-treated (n = 15), BMSCs + ES-treated (n = 15) and PBS-treated (n = 15). Dissociated BMSCs were resuspended at a concentration of 5 × 105 cells
per microliter. Rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP), and the injury site was exposed. The suspension of BMSCs or PBS was injected into the spinal cord as reported [5]. For electrodes implantation, the sterile electrodes were placed under the laminae of T9 and T11 with the duramatter intact. The electrodes were sutured to laminae with nonabsorbable sutures. The field strength was set as 0.4 mV/mm (39 mA/h, at 20 Hz), and lasted for 15 min twice a day. Another group of normal rats were set for control to check the safety of ES therapy which received only electrodes implantation and stimulation. Functional tests were performed before transplantation and weekly after transplantation. Locomotor activity was evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale for 4 min. Two independent blinded examiners observed hindlimb movements and assessed the animal’s locomotor function. The score was obtained by averaging the values of both limbs. Somatosensory evoked potentials (SSEPs) were measured before injury, after injury and weekly after BMSCs transplantation. A single square pulse of electrical stimulation was delivered with 0.2-ms pulse duration of stimuli and 1 mA intensity at 2 Hz. The latency of the evoked potentials was measured from the peak of the positive deflection (P1 latency). The amplitude was measured from the peak of the first negative deflection to the peak of the positive deflection (peak to peak amplitude). At the end of 8th week after injury, animals were anesthetized with 10% chloral hydrate; 4% paraformaldehyde was perfused intracardially for each rat. The whole spinal cord was removed postfixed in 4% paraformaldehyde overnight and half-cut longitudinally in the sagittal direction. Then sections from BMSCs-treated and BMSCs + ES-treated group were immunostained for BrdU and examined under microscope (CK40, Olympus, Japan) at a magnification of 400× for BMSCs survival.
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Table 1 Somatosensory evoked potentials 8th weeks after SCI. MSC SSEP 3w 4w 5w 6w 7w 8w *
PL (ms) 63.1 ± 2.2 54.3 ± 2.1 41.8 ± 0.9 36.7 ± 3.1 25.5 ± 1.6 23.1 ± 2.1
ES PA (v) 1.21 ± 0.10 2.03 ± 0.32 2.46 ± 0.12 2.65 ± 0.21 3.20 ± 0.11 3.48 ± 0.10
PL (ms) 65.3 ± 1.0 56.9 ± 2.7 42.7 ± 2.1 37.6 ± 2.6 26.5 ± 2.2 25.1 ± 1.0
MSC+ES PA (v) 1.10 ± 0.18 1.78 ± 0.21 2.43 ± 0.12 2.56 ± 0.22 3.05 ± 0.17 3.37 ± 0.16
PL (ms) 54.3 ± 2.5 47.7 ± 1.2 38.0 ± 1.5 28.3 ± 2.6 17.3 ± 1.2 12.8 ± 2.1*
PBS PA (v) 1.58 ± 0.32 2.53 ± 0.13 3.10 ± 0.15 3.98 ± 0.19 5.12 ± 0.11 5.68 ± 0.18*
PL(ms) – 63.8 ± 2.0 57.6 ± 1.3 54.3 ± 1.6 53.4 ± 2.7 51.2 ± 2.4
PA (v) – 1.36 ± 0.13 1.30 ± 0.18 1.75 ± 0.13 1.90 ± 0.21 1.85 ± 0.11
p < 0.01 compared to all other three groups.
A surviving cell count was conducted in 10 transect spinal cord sections within 10 mm around the spinal cord lesion site by morphometric image analysis (integrated optical density, IOD) with the use of ImageJ software (National Institute of Health). The number of surviving BMSCs was counted in 3 randomly selected unit areas (0.09 mm2 ), delineated by a calibrated reticle eyepiece. The average number and ratio of BrdU-labeled cells were used for comparison. Diffusion tensor imaging (DTI) was performed at 8th week after injury. Imaging involved the use of a 3T MRI system (GE EXCITE II, Wisconsin, USA) with a maximum gradient strength of 40 mT/m. A sequence of axial sensitivity encoding echo-planar DTI (field of view 270 mm × 270 mm; TR/TE 6000/83 ms; 4 m thick) was obtained [27]. The acquisition time of DTI was 5 min per rat. The fractional anisotropy (FA) is an anisotropic parameter: values closer to 1 represent a more anisotropic structure. The use of fibre track (FT) three-dimensional reconstructions can track the white matter pathways in the spinal cord. FA maps were generated and threedimensional FT maps were reconstructed at GEAW 2.0 workstation, the area of high anisotropy region (red color) was measured with the use of ImageJ software. The data analysis was through SPSS version 13.0. The data were analysed using one-way ANOVA. If equal variances were found, Fisher’s least significant difference test was performed. Otherwise, the Kruskal–Wallis Test and Dunnett’s T3 were used. The statistical significance level was set at p < 0.05 The Brdu-positive BMSCs were maily found around the cavity lesion of the spinal cord. The cells were well integrated in the host tissue in longitudinal sections (Fig. 1a and b). Furthermore, the number of surviving BMSCs in the BMSCs + ES group was significantly higher than that of the BMSCs group (147 ± 23 vs. 115 ± 16, p < 0.01, Fig. 1c). After calculating with the number of background cells, the ratio of BMSCs was still significantly higher in BMSCs + ES group (31.0 ± 4.6% vs. 26.8 ± 2.5%, p < 0.05, Fig. 1d), which further indicated an improved survival rate of BMSCs caused by ES. The longitudinal images of the leison area of the four groups at 8th week after injury are also presented (in supplemantary data). It is obvious that the area of cavity caused by injury was reduced in all three active treated group. The improvement was most remarkable in BMSCs + ES-treated group, with the regenerated tissue almost filling the cavity formed after injury. Behavior performance was evaluated in all rats to 8th week after injury. No major complication was observed in the normal control group set for ES control, which confirmed the safety of the implanted ES procedure (in supplemantary data). All rats underwent SCI lost hindlimbs function immediately after injury characterized by moving forward with forelimbs and instep placement of hindlimbs with no weight support. All rats in active treated groups exhibited a gradual improvement over time starting from 2nd week by recovering pain withdrawal reflex elicited by a controlled brief pinch. The recovery sped up obviously after 6th week, especially in BMSCs + ES-treated group (Fig. 2). At the end of 8th week, the score of the BMSCs + ES-treated rats was significantly improved to 13.2 ± 1.15, whereas the function of
the PBS-treated animals reached a score of 4.66 ± 0.33 (p < 0.01). The former score indicated a gait pattern characterized by frequent weight-supported plantar steps and consistent forelimb and hindlimb coordination. BMSCs + ES-treated group also showed significant higher score compared with the BMSCs-treated and EStreated alone group after the 4th week, most notably at the 8th week following the injury (BMSCs-treated: 8.76 ± 0.77; ES-treated: 8.53 ± 0.41; p < 0.01). There was no significant difference between the scores of BMSCs-treated and ES-treated group (p > 0.05). SSEPs were measured before injury, after injury and every week after MSCs transplantation. Detectable waves appeared from 3rd week after injury in all three active treated groups, whereas the same phenomenon started from 4th week in PBS-treated group (Table 1). At the end of 8th week following SCI, BMSCs + ES treated rats showed significantly shortened P1 latencies compared with those of PBS-treated controls (p < 0.01). BMSCs + ES-treated rats also showed increased amplitudes of SSEPs compared with the PBS-treated group (p < 0.01). In addition, the latencies of SSEP were significantly shorter and the amplitudes were increased in the BMSCs + ES-treated group compared to the BMSCs or ES group alone (p < 0.01). There was no significant difference between the latencies and amplitudes of BMSCs-treated and ES-treated group (p > 0.05). DTI imaging was performed at 8th week after the injury. The representative FA maps are shown in Fig. 3. In FA map, the red region of cervical cord indicates high anisotropy and the blue region of cerebrospinal fluid surrounding the red region indicates high isotropy. In PBS-treated group, the red region was fading and disconnected, which suggested decreased anisotropy and disconnected spinal cord at the lesion site caused by axon degeneration. The FA map
Fig. 2. Locomotor behavioral assessment after cell transplantation and ES. The scores of the BMSCs + ES-treated rats increased to 13.2 ± 1.15 at 8th week, whereas the function of the PBS-treated animals was only 4.66 ± 0.33 and BMSCs-treated animals were 8.76 ± 0.77. *p < 0.01 compared to all other groups by one-way ANOVA.
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Fig. 3. Representative FA maps of four groups. The red region of the cervical cord indicates high anisotropy (arrows). The FA map of BMSCs + ES-treated rats demonstrated improved shape and area of red region than the other three groups, which indicated an improved diffusion tensor cylinder in space. (a) BMSCs-treated group; (b) ES-treated group; (c) BMSCs + ES-treated group; (d) PBS-treated group; (e) FA values of rats in four groups. *p < 0.05 compared to other three groups; (f) the area of high anisotropy (red color) region in four groups. *p < 0.05 compared to other three groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of BMSCs + ES-treated group demonstrated improved shape of high anisotropy region than the other three groups, which indicated an improved diffusion tensor cylinder in space and enhanced axonal regeneration. Statistical analysis showed significant improvements
in FA value and the area of high anisotropy region (red color) of BMSCs + ES-treated group compared to other three groups (p < 0.05, Fig. 3e and f). The FT map of BMSCs + ES-treated group was smooth and showed better intergrity compared to BMSCs-treated alone and
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PBS-treated rats, which also supports enhanced axon regeneraion in combine treatment group (in supplemantary data). Transplantation of cultured MSCs has been successfully used in repairing neural loss after SCI [24,5,6,8,19]. However, the application of MSCs for damaged CNS is restricted by two major drawbacks: the poor survival rate and low differentiation rate into neurons [20,18,29]. Although several neurotrophic factors such as NT-3 and granulocyte macrophage-colony stimulating factor (GMCSF) [30] have been demonstrated to promote the survival of BMSCs and induce BMSCs to differentiate into neurons in vitro and in vivo, the effect of these factors is not clear. More importantly, these neurotrophic factors cannot maintain long-term effect due to their relatively short half-life. ES is a traditional method for the treatment of SCI for many years. Electro-acupuncture, which is an important part of traditional Chinese medicine, also shares the mechanism with ES. But its application is restricted due to the recognition of different meridians. In addition, implantable ES is recognized as a hotspot for the development of ES therapy. Implantable oscillating field stimulation has been approved by FDA for human trials on SCI [21]. Here, we investigated the effect of implanted electrodes connected to SSP machine, which is an improved therapy of electro-acupuncture, on BMSCs transplantation after SCI. The safety of implantation was proved in a normal control group of rats, with no rats suffering any neural complications. Furthermore, it was found that locomotor function and SSEPs in the ES and BMSCs + ES group were significantly improved than that in the PBS group and BMSCs group respectively. This result indicated that implanted spike wave ES could exert therapeutic effects in the treatment of SCI which was in consistent with other ES methods and traditional electroacupuncture therapy [9,21]. The improvement of BBB scores and SSEPs in BMSCs + ES group compared to BMSCs group was supported by the increased number and ratio of BMSCs confirmed by BrdU labeling. This was also observed in previous trials combing the use of BMSCs and ES as well as traditional electro-acupuncture [28,22]. It is well established that EF is of great importance in controlling stem cell behavior. Like all other cells, endogenous ion flows represent a set of signals in stem cells, and endogenous hyperpolarization is a functional determinant of MSC differentiation in vitro [23]. Oscillatory fields have been found to increase neural stem cell viability, possibly through the enhancement of mass transport due to electrokinetically driven flow [16]. Furthermore, Yan et al. showed that electro-acupuncture could elevate NT-3 expression in the injured spinal cord, thus enhance survival and differentiation of BMSCs [28]. This has immediate clinical significance because it is the bottleneck that the differentiation of BMSCs into neurons is not satisfactory in vivo. However, this study mainly focused on the safety and preliminary therapeutic effects of implanted SSP electrodes, further studies would be designed to investigate the mechanisms about BMSCs differentiation influenced by spike wave ES. Axon degeneration is the main reason for functional impairment caused by SCI. In this study, decreased FA value and area of high anisotropy region in DTI in injured spinal cord suggested decreased anisotropy caused by degeneration after SCI in rats [27]. However, the significantly improved FA value as well as area and shape of high anisotropy region in BMSCs + ES-treated group compared to BMSCs-treated group confirmed the increased axonal regeneration with spike wave stimulation on BMSCs. The underlying mechanism of the combined treatment of BMSCs transplantation plus ES treatment in axonal regeneration is still not very clear. Apart from increasing NT-3 secretion, electro-acupuncture has been confirmed to reduce glial scar formation by downregulating fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs) expression [9]. BMSCs could also secrete bioactive factors (G-CSF, BDNF, VEGF, IL-6, et cl.) that inhibit scar-
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ring, inhibit apotosis and stimulate angiogenesis, which is referred as “trophic activity” [4]. In addition, it is proved that ES could guide BMSCs’ directional migration [15] deep into the SCI, functioning as an important bridge for axon regeneration and remyelination [1]. These evidences imply that there may be a synergistic effect of these two therapies accounting for improved axonal regeneration in combine treatment. This hypothesis could also explain the fact that combined therapy could stimulate the axonal growth into lesion site and reduce cavity formation, which was confirmed in histology sections in our study. However, more researches are needed to further investigate the mechanisms about this. In summary, our experiment has shown for the first time that an implanted spike wave ES is safe in rats. It could also promote BMSCs survival, stimulate axonal regeneration and improve hindlimb locomotion. Our results suggest a new therapeutic potential of the combination of BMSCs transplantation with implanted spike wave ES to treat spinal cord injury. Acknowledgements The study was supported by grant from the Department of Science & Technology of Shandong Province, China (Project No. 2004GG3202011) to Prof. Y.Z. Chen. The authors are grateful to Prof. X.L. Wang, Baylor College of Medicine, for helpful comments on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.01.009. References [1] Y. Akiyama, C. Radtke, J.D. Kocsis, Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells, J. Neurosci. 22 (2002) 6623–6630. ˚ [2] T. Amemori, P. Jendelová, K. Ruzicková, D. Arboleda, E. Syková, Cotransplantation of olfactory ensheathing glia and mesenchymal stromal cells does not have synergistic effects after spinal cord injury in the rat, Cytotherapy 12 (2010) 212–225. [3] C.A. Ariza, A.T. Fleury, C.J. Tormos, V. Petruk, S. Chawla, J. Oh, D.S. Sakaguchi, S.K. Mallapragada, The influence of electric fields on hippocampal neural progenitor cells, Stem Cell Rev. (2010), July 28 [Epub ahead of print]. [4] A.I. Caplan, Adult mesenchymal stem cells for tissue engineering versus regenerative medicine, J. Cell. Physiol. 213 (2007) 341–347. [5] S.R. Cho, Y.R. Kim, H.S. Kang, S.H. Yim, C.I. Park, Y.H. Min, B.H. Lee, J.C. Shin, J.B. Lim, Functional recovery after the transplantation of neurally differentiated mesenchymal stem cells derived from bone barrow in a rat model of spinal cord injury, Cell Transplant. 18 (2009) 1359–1368. [6] J.S. Cho, H.W. Park, S.K. Park, S. Roh, S.K. Kang, K.S. Paik, M.S. Chang, Transplantation of mesenchymal stem cells enhances axonal outgrowth and cell survival in an organotypic spinal cord slice culture, Neurosci. Lett. 454 (2009) 43–48. [7] C.B. Choi, Y.K. Cho, K.V. Prakash, B.K. Jee, C.W. Han, Y.K. Paik, H.Y. Kim, K.H. Lee, N. Chung, H.K. Rha, Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells, Biochem. Biophys. Res. Commun. 350 (2006) 138–146. [8] Y. Ding, Q. Yan, J.W. Ruan, Y.Q. Zhang, W.J. Li, Y.J. Zhang, Y. Li, H. Dong, Y.S. Zeng, Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cordtransected rats, BMC Neurosci. 10 (2009) 35. [9] Y. Ding, Q. Yan, J.W. Ruan, Y.Q. Zhang, W.J. Li, X. Zeng, S.F. Huang, Y.J. Zhang, S. Wang, H. Dong, Y.S. Zeng, Bone marrow mesenchymal stem cells and electroacupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats, Cell Transplant. (2010) [Epub ahead of print]. [10] M.T. Fitch, J. Silver, CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure, Exp. Neurol. 209 (2009) 294–301. [11] S. Hamid, R. Hayek, Role of electrical stimulation for rehabilitation and regeneration after spinal cord injury: an overview, Eur. Spine J. 17 (2008) 1256–1269. [12] R. Hu, J. Zhou, C. Luo, J. Lin, X. Wang, X. Li, X. Bian, Y. Li, Q. Wan, Y. Yu, H. Feng, Glial scar and neuroregeneration: histological, functional, and magnetic resonance imaging analysis in chronic spinal cord injury, J. Neurosurg. Spine 13 (2010) 169–180. [13] M. Hyodo, T. Kitade, A Guide to Silver Spike Point (SSP) Therapy, 2nd ed., The Silver Spike Point Therapy Study Group, Tokyo, 1979.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Implanted spike wave electric stimulation promotes survival of the bone marrow mesenchymal stem cells and functional recovery in the spinal cord injured rats Wenliang Wu a , Hua Zhao a , Bin Xie b , Haichun Liu a , Yunzhen Chen a,∗ , Guangjun Jiao a , Hongliang Wang a a b
Department of Trauma Surgery, Shandong University Qilu Hospital, Wenhuaxi Road 107, Jinan 250012, China Department of Spine Surgery, Weihai Hospital, China
a r t i c l e
i n f o
Article history: Received 29 October 2010 Received in revised form 27 December 2010 Accepted 4 January 2011 Keywords: Mesenchymal stem cells Electric field stimulation Spinal cord injury
a b s t r a c t Transplantation of bone marrow-derived mesenchymal stromal cells (BMSCs) into the injured spinal cord may provide therapeutic benefit, but its application is limited by their poor survival and low differentiation rate into neurons. Electrical stimulation (ES) has been reported to promote survival and differentiation of the BMSCs. Therefore we investigated whether implanted spike wave ES could improve survival of BMSCs after transplantation and result in functional improvement in animals with spinal cord injury. Our results showed that the number and ratio of survived BMSCs near the lesion site were significantly increased in the BMSCs + ES-treated group as compared to BMSCs transplantation or ES treatment alone group. Furthermore, results from BBB scales, SSEP and DTI demonstrated a significant improved functional recovery in the BMSCs + ES group. This indicated that implanted spike wave ES could promote the bioactivity of BMSCs and their survival. This represents a new therapeutic potential of the combination of BMSCs transplantation with implanted spike wave ES to treat spinal cord injury. © 2011 Elsevier Ireland Ltd. All rights reserved.
Severe spinal cord injury (SCI) usually results in long-lasting deficits, involving partial or complete paralysis and loss of sensation below injury level. These deficits result from neuronal damage, demyelination of axons and dysfunctional neruoglial cells, all of which the central nervous system (CNS) has a limited ability to repair itself [24]. The failure of axonal regeneration following SCI has also been attributed to a non-permissive environment containing inflammatory mediators, lack of neurotrophic support and formation of glial scars [10,12]. It has been suggested that transplantation of bone marrowderived mesenchymal stem cells (BMSCs) into the injured spinal cord may provide therapeutic benefit [24,5,6,8,19,20,30]. One possible mechanism is that BMSCs can differentiate into neural lineage cells under specific in vitro conditions [20,17,26,4]. However, these differentiated “neurons” are only identified to resemble true neurons phenotypically but different in electrophysiological characteristics [25,7]. Second, BMSCs secrete a broad spectrum of bioactive macromolecules that could be immunoregulatory and contributing to structure regenerative microenvironments in fields of injury [19,20,4]. Other possible mechanisms include remyelination and a bridging effect for axon regeneration [1]. Despite these
∗ Corresponding author. Tel.: +86 13606373827. E-mail address: [email protected] (Y. Chen). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.01.009
advantages, the application of BMSCs is limited by their poor survival and low differentiation rate into neurons when transplanted into injured spinal cord [20,7,2]. The use of electrical stimulation (ES) has been involved in restoring the lost functions following spinal cord injury for decades. Several therapeutic devices are commercially available at present, including devices for functional electrical stimulation (FES) [11], direct current stimulation, oscillating field stimulation (OFS) [21], and electro-acupuncture (EA) [8]. The common background for these therapies is the consensus that electrical fields (EFs) serve as important physiological signals and play a crucial role in the nervous system development and regeneration [11,14,23]. Numerous researches have also investigated the effect of ES on stem cells. Weak direct current (DC) has been confirmed to regulate stem/progenitor migration or differentiation [3,15]. Alternating current could also exert improved effects on neural stem cell viability and differentiation [16]. Furthermore, EA has been reported to promote survival and differentiation of the BMSCs in rats [8]. Thus, we hypothesize that maybe implanted electrodes releasing ES in vivo could have the same effects on BMSCs in vitro. Silver spike point therapy (SSP), which is also known as needlefree electro-acupuncture therapy, has been confirmed to regulate pain by generating a physiological spike wave with bi-directional symmetrical waveforms [13]. It is reasonable that the physiological low-frequency spike wave could also prolong BMSCs survival in
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Fig. 1. The survival of BMSCs in the injured spinal cord. BrdU-labeled (brown, red arrow) BMSCs were well integrated with host tissue. (a) BMSCs-treated group; (b) BMSCs + ES-treated group; (c) the number of survived BMSCs within 10 mm of the lesion site per unit area (**p < 0.01); (d) the ratio of survived BMSCs within 10 mm of the lesion site (*p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
injured spinal cord as EA did. To our knowledge, this combination use of BMSCs transplantation and implanted spike wave ES has not been investigated before. In this study, we first examined whether implanted spike wave ES could exert effects on BMSCs survival after transplantation in rats spinal cord injury model. Then we investigated whether this combined treatment resulted in any functional improvement than single treatment of each. Our results demonstrated that implanted physiological spike wave could prolong BMSCs survival and improve functional recovery after BMSCs transplantation. Following harvesting BM aspirates from the femur of adult male Sprague–Dawley rats (Animal Center of Shandong University Medical School, Jinan, China), mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lymphoprep, Fresenius Kabi Norge, AS) and passaged as routine. For immunohistological tests, cells were incubated with 10 mol/L 5-bromo-2-deoxyuridine (BrdU, Sigma–Aldrich, USA) for 3 days before transplantation. The device for ES was designed using s a multi-frequency SSP machine (Felicia TM21, Nihon Medix, Japan). The electrodes for implantation were made of standard pacemaker cables and plantinum/iridium tips with a 2-mm2 surface area. The spike wave stimulation generated was the same as that used in traditional EA. All protocols were approved by Shandong University Committee on Animal Research. Animals were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP). A laminectomy was performed at the T10 spinal cord level with duramatter intact. Contused incomplete SCI was induced by dropping a 10-g weight rod from a 6 cm height onto the exposed dorsal surface of the spinal cord using an NYU impactor. One week after SCI, the injured rats were randomly assigned to four groups: BMSCs-treated (n = 15), ES-treated (n = 15), BMSCs + ES-treated (n = 15) and PBS-treated (n = 15). Dissociated BMSCs were resuspended at a concentration of 5 × 105 cells
per microliter. Rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP), and the injury site was exposed. The suspension of BMSCs or PBS was injected into the spinal cord as reported [5]. For electrodes implantation, the sterile electrodes were placed under the laminae of T9 and T11 with the duramatter intact. The electrodes were sutured to laminae with nonabsorbable sutures. The field strength was set as 0.4 mV/mm (39 mA/h, at 20 Hz), and lasted for 15 min twice a day. Another group of normal rats were set for control to check the safety of ES therapy which received only electrodes implantation and stimulation. Functional tests were performed before transplantation and weekly after transplantation. Locomotor activity was evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale for 4 min. Two independent blinded examiners observed hindlimb movements and assessed the animal’s locomotor function. The score was obtained by averaging the values of both limbs. Somatosensory evoked potentials (SSEPs) were measured before injury, after injury and weekly after BMSCs transplantation. A single square pulse of electrical stimulation was delivered with 0.2-ms pulse duration of stimuli and 1 mA intensity at 2 Hz. The latency of the evoked potentials was measured from the peak of the positive deflection (P1 latency). The amplitude was measured from the peak of the first negative deflection to the peak of the positive deflection (peak to peak amplitude). At the end of 8th week after injury, animals were anesthetized with 10% chloral hydrate; 4% paraformaldehyde was perfused intracardially for each rat. The whole spinal cord was removed postfixed in 4% paraformaldehyde overnight and half-cut longitudinally in the sagittal direction. Then sections from BMSCs-treated and BMSCs + ES-treated group were immunostained for BrdU and examined under microscope (CK40, Olympus, Japan) at a magnification of 400× for BMSCs survival.
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Table 1 Somatosensory evoked potentials 8th weeks after SCI. MSC SSEP 3w 4w 5w 6w 7w 8w *
PL (ms) 63.1 ± 2.2 54.3 ± 2.1 41.8 ± 0.9 36.7 ± 3.1 25.5 ± 1.6 23.1 ± 2.1
ES PA (v) 1.21 ± 0.10 2.03 ± 0.32 2.46 ± 0.12 2.65 ± 0.21 3.20 ± 0.11 3.48 ± 0.10
PL (ms) 65.3 ± 1.0 56.9 ± 2.7 42.7 ± 2.1 37.6 ± 2.6 26.5 ± 2.2 25.1 ± 1.0
MSC+ES PA (v) 1.10 ± 0.18 1.78 ± 0.21 2.43 ± 0.12 2.56 ± 0.22 3.05 ± 0.17 3.37 ± 0.16
PL (ms) 54.3 ± 2.5 47.7 ± 1.2 38.0 ± 1.5 28.3 ± 2.6 17.3 ± 1.2 12.8 ± 2.1*
PBS PA (v) 1.58 ± 0.32 2.53 ± 0.13 3.10 ± 0.15 3.98 ± 0.19 5.12 ± 0.11 5.68 ± 0.18*
PL(ms) – 63.8 ± 2.0 57.6 ± 1.3 54.3 ± 1.6 53.4 ± 2.7 51.2 ± 2.4
PA (v) – 1.36 ± 0.13 1.30 ± 0.18 1.75 ± 0.13 1.90 ± 0.21 1.85 ± 0.11
p < 0.01 compared to all other three groups.
A surviving cell count was conducted in 10 transect spinal cord sections within 10 mm around the spinal cord lesion site by morphometric image analysis (integrated optical density, IOD) with the use of ImageJ software (National Institute of Health). The number of surviving BMSCs was counted in 3 randomly selected unit areas (0.09 mm2 ), delineated by a calibrated reticle eyepiece. The average number and ratio of BrdU-labeled cells were used for comparison. Diffusion tensor imaging (DTI) was performed at 8th week after injury. Imaging involved the use of a 3T MRI system (GE EXCITE II, Wisconsin, USA) with a maximum gradient strength of 40 mT/m. A sequence of axial sensitivity encoding echo-planar DTI (field of view 270 mm × 270 mm; TR/TE 6000/83 ms; 4 m thick) was obtained [27]. The acquisition time of DTI was 5 min per rat. The fractional anisotropy (FA) is an anisotropic parameter: values closer to 1 represent a more anisotropic structure. The use of fibre track (FT) three-dimensional reconstructions can track the white matter pathways in the spinal cord. FA maps were generated and threedimensional FT maps were reconstructed at GEAW 2.0 workstation, the area of high anisotropy region (red color) was measured with the use of ImageJ software. The data analysis was through SPSS version 13.0. The data were analysed using one-way ANOVA. If equal variances were found, Fisher’s least significant difference test was performed. Otherwise, the Kruskal–Wallis Test and Dunnett’s T3 were used. The statistical significance level was set at p < 0.05 The Brdu-positive BMSCs were maily found around the cavity lesion of the spinal cord. The cells were well integrated in the host tissue in longitudinal sections (Fig. 1a and b). Furthermore, the number of surviving BMSCs in the BMSCs + ES group was significantly higher than that of the BMSCs group (147 ± 23 vs. 115 ± 16, p < 0.01, Fig. 1c). After calculating with the number of background cells, the ratio of BMSCs was still significantly higher in BMSCs + ES group (31.0 ± 4.6% vs. 26.8 ± 2.5%, p < 0.05, Fig. 1d), which further indicated an improved survival rate of BMSCs caused by ES. The longitudinal images of the leison area of the four groups at 8th week after injury are also presented (in supplemantary data). It is obvious that the area of cavity caused by injury was reduced in all three active treated group. The improvement was most remarkable in BMSCs + ES-treated group, with the regenerated tissue almost filling the cavity formed after injury. Behavior performance was evaluated in all rats to 8th week after injury. No major complication was observed in the normal control group set for ES control, which confirmed the safety of the implanted ES procedure (in supplemantary data). All rats underwent SCI lost hindlimbs function immediately after injury characterized by moving forward with forelimbs and instep placement of hindlimbs with no weight support. All rats in active treated groups exhibited a gradual improvement over time starting from 2nd week by recovering pain withdrawal reflex elicited by a controlled brief pinch. The recovery sped up obviously after 6th week, especially in BMSCs + ES-treated group (Fig. 2). At the end of 8th week, the score of the BMSCs + ES-treated rats was significantly improved to 13.2 ± 1.15, whereas the function of
the PBS-treated animals reached a score of 4.66 ± 0.33 (p < 0.01). The former score indicated a gait pattern characterized by frequent weight-supported plantar steps and consistent forelimb and hindlimb coordination. BMSCs + ES-treated group also showed significant higher score compared with the BMSCs-treated and EStreated alone group after the 4th week, most notably at the 8th week following the injury (BMSCs-treated: 8.76 ± 0.77; ES-treated: 8.53 ± 0.41; p < 0.01). There was no significant difference between the scores of BMSCs-treated and ES-treated group (p > 0.05). SSEPs were measured before injury, after injury and every week after MSCs transplantation. Detectable waves appeared from 3rd week after injury in all three active treated groups, whereas the same phenomenon started from 4th week in PBS-treated group (Table 1). At the end of 8th week following SCI, BMSCs + ES treated rats showed significantly shortened P1 latencies compared with those of PBS-treated controls (p < 0.01). BMSCs + ES-treated rats also showed increased amplitudes of SSEPs compared with the PBS-treated group (p < 0.01). In addition, the latencies of SSEP were significantly shorter and the amplitudes were increased in the BMSCs + ES-treated group compared to the BMSCs or ES group alone (p < 0.01). There was no significant difference between the latencies and amplitudes of BMSCs-treated and ES-treated group (p > 0.05). DTI imaging was performed at 8th week after the injury. The representative FA maps are shown in Fig. 3. In FA map, the red region of cervical cord indicates high anisotropy and the blue region of cerebrospinal fluid surrounding the red region indicates high isotropy. In PBS-treated group, the red region was fading and disconnected, which suggested decreased anisotropy and disconnected spinal cord at the lesion site caused by axon degeneration. The FA map
Fig. 2. Locomotor behavioral assessment after cell transplantation and ES. The scores of the BMSCs + ES-treated rats increased to 13.2 ± 1.15 at 8th week, whereas the function of the PBS-treated animals was only 4.66 ± 0.33 and BMSCs-treated animals were 8.76 ± 0.77. *p < 0.01 compared to all other groups by one-way ANOVA.
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Fig. 3. Representative FA maps of four groups. The red region of the cervical cord indicates high anisotropy (arrows). The FA map of BMSCs + ES-treated rats demonstrated improved shape and area of red region than the other three groups, which indicated an improved diffusion tensor cylinder in space. (a) BMSCs-treated group; (b) ES-treated group; (c) BMSCs + ES-treated group; (d) PBS-treated group; (e) FA values of rats in four groups. *p < 0.05 compared to other three groups; (f) the area of high anisotropy (red color) region in four groups. *p < 0.05 compared to other three groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
of BMSCs + ES-treated group demonstrated improved shape of high anisotropy region than the other three groups, which indicated an improved diffusion tensor cylinder in space and enhanced axonal regeneration. Statistical analysis showed significant improvements
in FA value and the area of high anisotropy region (red color) of BMSCs + ES-treated group compared to other three groups (p < 0.05, Fig. 3e and f). The FT map of BMSCs + ES-treated group was smooth and showed better intergrity compared to BMSCs-treated alone and
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PBS-treated rats, which also supports enhanced axon regeneraion in combine treatment group (in supplemantary data). Transplantation of cultured MSCs has been successfully used in repairing neural loss after SCI [24,5,6,8,19]. However, the application of MSCs for damaged CNS is restricted by two major drawbacks: the poor survival rate and low differentiation rate into neurons [20,18,29]. Although several neurotrophic factors such as NT-3 and granulocyte macrophage-colony stimulating factor (GMCSF) [30] have been demonstrated to promote the survival of BMSCs and induce BMSCs to differentiate into neurons in vitro and in vivo, the effect of these factors is not clear. More importantly, these neurotrophic factors cannot maintain long-term effect due to their relatively short half-life. ES is a traditional method for the treatment of SCI for many years. Electro-acupuncture, which is an important part of traditional Chinese medicine, also shares the mechanism with ES. But its application is restricted due to the recognition of different meridians. In addition, implantable ES is recognized as a hotspot for the development of ES therapy. Implantable oscillating field stimulation has been approved by FDA for human trials on SCI [21]. Here, we investigated the effect of implanted electrodes connected to SSP machine, which is an improved therapy of electro-acupuncture, on BMSCs transplantation after SCI. The safety of implantation was proved in a normal control group of rats, with no rats suffering any neural complications. Furthermore, it was found that locomotor function and SSEPs in the ES and BMSCs + ES group were significantly improved than that in the PBS group and BMSCs group respectively. This result indicated that implanted spike wave ES could exert therapeutic effects in the treatment of SCI which was in consistent with other ES methods and traditional electroacupuncture therapy [9,21]. The improvement of BBB scores and SSEPs in BMSCs + ES group compared to BMSCs group was supported by the increased number and ratio of BMSCs confirmed by BrdU labeling. This was also observed in previous trials combing the use of BMSCs and ES as well as traditional electro-acupuncture [28,22]. It is well established that EF is of great importance in controlling stem cell behavior. Like all other cells, endogenous ion flows represent a set of signals in stem cells, and endogenous hyperpolarization is a functional determinant of MSC differentiation in vitro [23]. Oscillatory fields have been found to increase neural stem cell viability, possibly through the enhancement of mass transport due to electrokinetically driven flow [16]. Furthermore, Yan et al. showed that electro-acupuncture could elevate NT-3 expression in the injured spinal cord, thus enhance survival and differentiation of BMSCs [28]. This has immediate clinical significance because it is the bottleneck that the differentiation of BMSCs into neurons is not satisfactory in vivo. However, this study mainly focused on the safety and preliminary therapeutic effects of implanted SSP electrodes, further studies would be designed to investigate the mechanisms about BMSCs differentiation influenced by spike wave ES. Axon degeneration is the main reason for functional impairment caused by SCI. In this study, decreased FA value and area of high anisotropy region in DTI in injured spinal cord suggested decreased anisotropy caused by degeneration after SCI in rats [27]. However, the significantly improved FA value as well as area and shape of high anisotropy region in BMSCs + ES-treated group compared to BMSCs-treated group confirmed the increased axonal regeneration with spike wave stimulation on BMSCs. The underlying mechanism of the combined treatment of BMSCs transplantation plus ES treatment in axonal regeneration is still not very clear. Apart from increasing NT-3 secretion, electro-acupuncture has been confirmed to reduce glial scar formation by downregulating fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs) expression [9]. BMSCs could also secrete bioactive factors (G-CSF, BDNF, VEGF, IL-6, et cl.) that inhibit scar-
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ring, inhibit apotosis and stimulate angiogenesis, which is referred as “trophic activity” [4]. In addition, it is proved that ES could guide BMSCs’ directional migration [15] deep into the SCI, functioning as an important bridge for axon regeneration and remyelination [1]. These evidences imply that there may be a synergistic effect of these two therapies accounting for improved axonal regeneration in combine treatment. This hypothesis could also explain the fact that combined therapy could stimulate the axonal growth into lesion site and reduce cavity formation, which was confirmed in histology sections in our study. However, more researches are needed to further investigate the mechanisms about this. In summary, our experiment has shown for the first time that an implanted spike wave ES is safe in rats. It could also promote BMSCs survival, stimulate axonal regeneration and improve hindlimb locomotion. Our results suggest a new therapeutic potential of the combination of BMSCs transplantation with implanted spike wave ES to treat spinal cord injury. Acknowledgements The study was supported by grant from the Department of Science & Technology of Shandong Province, China (Project No. 2004GG3202011) to Prof. Y.Z. Chen. The authors are grateful to Prof. X.L. Wang, Baylor College of Medicine, for helpful comments on the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.01.009. References [1] Y. Akiyama, C. Radtke, J.D. Kocsis, Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells, J. Neurosci. 22 (2002) 6623–6630. ˚ [2] T. Amemori, P. Jendelová, K. Ruzicková, D. Arboleda, E. Syková, Cotransplantation of olfactory ensheathing glia and mesenchymal stromal cells does not have synergistic effects after spinal cord injury in the rat, Cytotherapy 12 (2010) 212–225. [3] C.A. Ariza, A.T. Fleury, C.J. Tormos, V. Petruk, S. Chawla, J. Oh, D.S. Sakaguchi, S.K. Mallapragada, The influence of electric fields on hippocampal neural progenitor cells, Stem Cell Rev. (2010), July 28 [Epub ahead of print]. [4] A.I. Caplan, Adult mesenchymal stem cells for tissue engineering versus regenerative medicine, J. Cell. Physiol. 213 (2007) 341–347. [5] S.R. Cho, Y.R. Kim, H.S. Kang, S.H. Yim, C.I. Park, Y.H. Min, B.H. Lee, J.C. Shin, J.B. Lim, Functional recovery after the transplantation of neurally differentiated mesenchymal stem cells derived from bone barrow in a rat model of spinal cord injury, Cell Transplant. 18 (2009) 1359–1368. [6] J.S. Cho, H.W. Park, S.K. Park, S. Roh, S.K. Kang, K.S. Paik, M.S. Chang, Transplantation of mesenchymal stem cells enhances axonal outgrowth and cell survival in an organotypic spinal cord slice culture, Neurosci. Lett. 454 (2009) 43–48. [7] C.B. Choi, Y.K. Cho, K.V. Prakash, B.K. Jee, C.W. Han, Y.K. Paik, H.Y. Kim, K.H. Lee, N. Chung, H.K. Rha, Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells, Biochem. Biophys. Res. Commun. 350 (2006) 138–146. [8] Y. Ding, Q. Yan, J.W. Ruan, Y.Q. Zhang, W.J. Li, Y.J. Zhang, Y. Li, H. Dong, Y.S. Zeng, Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cordtransected rats, BMC Neurosci. 10 (2009) 35. [9] Y. Ding, Q. Yan, J.W. Ruan, Y.Q. Zhang, W.J. Li, X. Zeng, S.F. Huang, Y.J. Zhang, S. Wang, H. Dong, Y.S. Zeng, Bone marrow mesenchymal stem cells and electroacupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats, Cell Transplant. (2010) [Epub ahead of print]. [10] M.T. Fitch, J. Silver, CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure, Exp. Neurol. 209 (2009) 294–301. [11] S. Hamid, R. Hayek, Role of electrical stimulation for rehabilitation and regeneration after spinal cord injury: an overview, Eur. Spine J. 17 (2008) 1256–1269. [12] R. Hu, J. Zhou, C. Luo, J. Lin, X. Wang, X. Li, X. Bian, Y. Li, Q. Wan, Y. Yu, H. Feng, Glial scar and neuroregeneration: histological, functional, and magnetic resonance imaging analysis in chronic spinal cord injury, J. Neurosurg. Spine 13 (2010) 169–180. [13] M. Hyodo, T. Kitade, A Guide to Silver Spike Point (SSP) Therapy, 2nd ed., The Silver Spike Point Therapy Study Group, Tokyo, 1979.
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