Electro‐acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment: A summary of current laboratory findings and a review of literature

Abstract The incidence and disability rate of spinal cord injury (SCI) worldwide are high, imposing a heavy burden on patients. Considerable research efforts have been directed toward identifying new strategies to effectively treat SCI. Governor Vessel electro‐acupuncture (GV‐EA), used in traditional Chinese medicine, combines acupuncture with modern electrical stimulation. It has been shown to improve the microenvironment of injured spinal cord (SC) by increasing levels of endogenous neurotrophic factors and reducing inflammation, thereby protecting injured neurons and promoting myelination. In addition, axons extending from transplanted stem cell‐derived neurons can potentially bridge the two severed ends of tissues in a transected SC to rebuild neuronal circuits and restore motor and sensory functions. However, every single treatment approach to severe SCI has proven unsatisfactory. Combining different treatments—for example, electro‐acupuncture (EA) with adult stem cell transplantation—appears to be a more promising strategy. In this review, we have summarized the recent progress over the past two decades by our team especially in the use of GV‐EA for the repair of SCI. By this strategy, we have shown that EA can stimulate the nerve endings of the meningeal branch. This would elicit the dorsal root ganglion neurons to secrete excess amounts of calcitonin gene‐related peptide centrally in the SC. The neuropeptide then activates the local cells to secrete neurotrophin‐3 (NT‐3), which mediates the survival and differentiation of donor stem cells overexpressing the NT‐3 receptor, at the injury/graft site of the SC. Increased local production of NT‐3 facilitates reconstruction of host neural tissue such as nerve fiber regeneration and myelination. All this events in sequence would ultimately strengthen the cortical motor‐evoked potentials and restore the motor function of paralyzed limbs. The information presented herein provides a basis for future studies on the clinical application of GV‐EA and adult stem cell transplantation for the treatment of SCI.


| INTRODUC TI ON
There is presently no effective treatment for severe spinal cord injury (SCI) because the microenvironment of the injured spinal cord (SC) tissue is not conducive to self-repair of disrupted neural pathways. 1 As such, improving the microenvironment of injured SC tissue, so that it promotes tissue repair, has been the focus of intense research. 2 Neurotrophic factors that have been used for this purpose include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT-3). 3 Both NGF and its receptor, tyrosine kinase (Trk)A, mainly act on sensory neurons, 4 with no obvious effects on motor neurons. BDNF and its receptor, TrkB, act on a limited number of neuron types. 5 NT-3 and its receptor, TrkC, play an important role in the development and differentiation of neurons, as well as in the survival and axonal regeneration of damaged central neurons (in the brain and SC). 6 Furthermore, NT-3 has also been shown to promote the regeneration of corticospinal tract nerve fibers after SCI, 7 although the effect is not maintained over a long period. To address this problem, our research team has adopted a neuromodulation technique, based on traditional Chinese medicine acupuncture combined with modern electrical stimulation (EA) to trigger the continuous secretion of endogenous NT-3 in the SC.
In traditional Chinese medicine, paraplegia caused by SCI is categorized as an "indolence," "wind," and "sputum" syndrome and is thought to result from damage to the Governor Vessel (GV), 8 which regulates the function of all yang medians. SCI is associated with GV damage, which prevents the flow of qi and blood, leading to atrophy and loss of muscle function. 9 In theory, EA at the GV acts directly on a diseased area such as the injured SC to replenish the yang medians and increase their accessibility, thereby alleviating paraplegia.
Moreover, EA at the GV acupoints (located at the posterior midline of the trunk) is the preferred treatment for paraplegia caused by SCI. [8][9][10][11][12] A key concept of GV-EA is that stimulation at specific somatic tissues/acupoints can modulate internal SC physiology. In GV-EA, a small low-frequency pulsed current is delivered through needles inserted at the GV acupoints, which ventilates the meridians to promote qi and blood flow. 8 Thus, this therapy has the dual effects of acupuncture and electrical stimulation. Furthermore, GV-EA has been shown to improve traumatic paraplegia. 13 Some aspects of the mechanism underlying this effect have been elucidated over the last two decades. Both EA and GV-EA can alleviate secondary damage after SCI, protect neurons, and stimulate nerve fiber regeneration and functional recovery by regulating the levels of neurotrophic factors, neurotransmitters, neuropeptides, and intracellular signaling pathway components. [14][15][16][17] Our previous work has demonstrated that GV-EA improves the microenvironment of the injured SC and promotes the regeneration of damaged nerve fibers and functional recovery. 18 EA was shown to stimulate the migration, proliferation, and differentiation of endogenous neural stem cells (NSCs) at the injury site in the SC of rats. [19][20][21] Under normal conditions, these NSCs are induced to differentiate into astrocytes by factors in the microenvironment 22 that participate in the formation of glial scars for tissue stabilization and repair.
NSCs rarely differentiate into neurons to replace those that have died due to injury; therefore, whereas GV-EA can promote SC repair, the depletion of endogenous NSCs, or their insufficient differentiation into neurons, prevents the restoration of neuronal circuits in the absence of any transplantation of exogenous NSCs. 23 The transplantation of NSCs into the injured SC is a cell-based therapy that has been used for more than two decades. 1 When embryonic neural tissue is transplanted into the developing or adult SC, NSCs or newborn neurons replace lost nerve cells, contributing to the reconstruction of neuronal circuits and functional recovery. 24 NSCs can be induced to differentiate into neurons, astrocytes, and oligodendrocytes under appropriate conditions. NSCs for transplantation are mainly derived from embryonic and adult tissues.
Adult NSCs repair SC injury by replacing dead neurons and thus facilitate the reconstruction of neuronal circuits 25 ; the myelination of regenerated nerve fibers, 26 and secreting neurotrophic factors to improve the microenvironment of the injured SC and promote nerve fiber regeneration. 27 Bone marrow mesenchymal stem cells (MSCs)-another type of adult stem cell that can be isolated from a variety of sources-rapidly proliferate in vitro and exhibit multidirectional differentiation potential. When transplanted into the body, MSCs can be induced to differentiate into chondroblasts, osteoblasts, adipocytes, myoblasts, cardiomyocytes, hepatocytes, oligodendrocytes, and neuron-like cells by factors in the local microenvironment. 28 It is apparent from the literature that there are some critical issues and inherent limitations that need to be fully addressed whether we are to treat SCI more effectively. We consider the following strategies crucial for the better treatment of SCI: (1) to improve the K E Y W O R D S bone marrow mesenchymal stem cells, electrical stimulation, electro-acupuncture, neuromodulation, neurotrophic factors, receptor tyrosine kinases; neural stem cells, spinal cord injury microenvironment of the damaged tissue so it is conducive for the enhancement of nerve regeneration after SCI; (2) to replace the dead neurons, oligodendrocytes, and other major functional cells, and (3) to promote the better integration of replacement functional cells into the original neural network of the SC. Collectively, these factors will eventually help to repair the damaged structure and restore function in the SC. In this review, we summarize the research conducted over the last two decades relating to the therapeutic potential of GV-EA in combination with adult stem cell transplantation for the repair of the injured SC, paying particular focus upon the data generated by our laboratory.

| GV-E APROMOTE STHESURVIVALOF INJ UREDSCNEURON S
The application of acupuncture and EA for the treatment of nervous system disorders has become widely accepted over recent years, particularly as the mechanistic basis of their effects have been elucidated. 30 Recent research revealed that the neuroprotection provided by EA was associated with activation of the parasympathetic nervous system in experimental stroke model animals. 31 EA pretreatment also can increase ambient endocannabinoid levels and result in activation of the ischemic penumbral astroglial cannabinoid type 1 receptor, which led to moderate upregulation of extracellular glutamate that protected neurons from cerebral ischemic injury. 32 In addition, EA can promote the survival and synaptic plasticity of hippocampal neurons and improve spatial memory disorders caused by sleep deprivation. 33 Interestingly, a previous research suggested that EA protected cerebral hippocampal neurons in vascular dementia by inhibiting the expression of p53 (a tumor suppressor) and Noxa (p53 downstream effector) in hippocampal CA1 region. 34 In response to EA, neurons synthesize and secrete specific proteins and neuropeptides that transduce the electrical signals. At different frequencies, EA can reportedly cause the central nervous system to release different types of neuropeptides that promote tissue recovery. 35 Neuropeptides are a class of neurotransmitters or neuromodulators that are characterized by slow induction and prolonged action in a manner that is comparable to the slow conduction of EA needles.
The SC receives direct peripheral sensory input from the trunk and limbs, with peptidergic nerve fibers projecting to the dorsal horn. The fiber endings localized at the acupoints sense the needle and then release peptide substances (e.g., substance P [SP], vasoactive intestinal peptide [VIP], and neuropeptide [NP]Y). 36 Research suggests that GV-EA causes cells in the damaged SC to secrete acupoint-specific proteins and neuropeptides that promote neuronal survival. In our laboratory, we found that GV-EA increases the expression of acupoint-specific proteins, annexin (ANX)A5, and collapsin response-mediated protein (CRMP)2 in the injured SC tissue of rats. 16 Moreover, the application of EA at non-acupoints, and at the Huatuo-jiaji acupoint, has been shown to downregulate these proteins.
In addition, ANXA5 and CRMP2 have been shown to exert protective effects on injured neurons in the SC nucleus dorsalis. 16 In other studies, we showed that GV-EA, EA at non-acupoints, and EA at the Huatuo-jiaji acupoint, has distinct effects on calcitonin generelated peptide (CGRP) levels in the injured SC tissue of rats; the application of GV-EA increased these expression levels. 37,38 Moreover, CGRP promotes the survival of injured neurons in the nucleus dorsalis and red nucleus of the midbrain. At the Huatuo-jiaji acupoint, GV-EA was also found to be more effective than EA in reducing the expression of nitric oxide synthase in injured neurons of the nucleus dorsalis, thus enhancing neuronal survival. 39 These findings provide evidence that EA can stimulate afferent nerve fibers in GV acupoints to induce the synthesis and secretion of specific proteins or neuropeptides by SC neurons to promote the survival of injured neurons.

| GV-E AS TIMUL ATE STHESCVIA AFFERENTNERVEFIB ER SOFTHE MENING E ALB R AN CH
The use of GV-EA for the treatment of SCI in rats is generally performed at acupuncture points below the spinous processes of the vertebral column corresponding to the upper and lower segments of the injury site (at an insertion depth of about 5 mm that is achieved by twirling, Figure 1). The positive and negative electrodes deliver a low-frequency electrical pulse through upper and lower 0.30-mm diameter needles. 37 In our previous work, stimulation was performed with a sparse-dense wave alternating stimulation pulse (alternating between 50 Hz/1.05 s and 2 Hz/2.85 s, with a pulse width of 0.5 ms), at a current intensity of approximately 5 µA between the two acupoints flanking the lesion site (the intensity was determined based on the observation of a slight vibration of the rat's muscle); the retention time was 20 min, and the frequency was once every other day. 40 In our previous studies, we speculated that GV-EA acts directly on the meningeal branch and perhaps also the dorsal (posterior) ramus of the spinal nerve of the SC. The acupuncture needle that penetrates the acupoint should pass through the skin, supraspinous ligament, interspinous ligament, and ligamentum flavum. It has been reported that the posterior longitudinal ligament of the vertebral bodies is also innervated by nerve endings of the meningeal branch. 41 The meningeal branch contains the sensory afferent nerve fibers from the dorsal root ganglion (DRG) and some sympathetic nerve fibers originating from the adjacent paravertebral ganglia. 41 We hypothesized that electrical stimulation would activate the GV acupoint to produce local sensory signals that are conveyed to the SC through afferent nerve fibers. 40 To test this hypothesis, we injected red fluorescent cholera toxin B subunit CTB-555 into the GV acupoint region to mark af-

| GV-E AINDUCE STHESYNTHE S ISAND S ECRE TI ONOFNEUROTROPHI CFAC TOR S BYCELL SINTHEINJ UREDSC
Following SC transection and demyelination, cells in the injured area and adjacent tissue undergo a number of pathophysiological changes, including the reduced synthesis and secretion of neurotrophic factors; this is one of the main factors underlying the inhibition of nerve repair. An important therapeutic mechanism of GV-EA in SCI is to induce cells in the SC to synthesize and secrete endogenous neurotrophic factors, 43 which creates a microenvironment that is conducive to neuronal survival and axonal regeneration. 14,44,45 NT-3 is a neurotrophic factor that plays an important role in preventing the death of injured neurons, enhancing neuronal survival and axonal regeneration, and inducing endogenous oligodendrocyte precursor cells to differentiate into mature oligodendrocytes that can restore the myelin sheath. 46 GV-EA has been shown to stimulate the synthesis and secretion of NT-3 by various cell types in transected SC tissue, [47][48][49] including neurons, astrocytes, microglia/ macrophages, and oligodendrocytes. The secretion of NT-3 by these cells progressively increases from Day 1 through 7 of GV-EA application; the level on Day 7 is significantly higher than that prior to the application of EA.. 47 We also found that NT-3 levels were M2-type microglia/macrophages, thus reducing the inflammatory response. [60][61][62][63] However, the exact mechanisms by which NT-3 inhibits the inflammatory response for SCI repair have yet to be elucidated.

| GV-E APROMOTE SNT-3S ECRE TI ON INTHEINJ UREDSCTOALLE VIATEMUSCLE ATROPHYINPAR ALY ZEDLIMBS
Studies have shown that sensory and motor functions are lost below the level of injury following severe SCI. 64 Due to the loss of innervation, the volume and weight of the muscle in the paralyzed limbs are reduced as the muscles undergo atrophy, 65,66 thus further hindering the recovery of motor function. Therefore, maintaining muscle function is essential for functional recovery from SC injury. 67 Although the ascending and descending neural pathways are transected in a thoracic segment SCI, below the injury level, the structural basis of the neural circuits that regulate the lower limbs remain in the lumbar segment of the SC. [68][69][70] These neural circuits may be functionally silent, and their reactivation can promote the nerve-muscle connection and prevent muscle atrophy. [65][66][67][68][69][70] We applied GV-EA to rats with a completely transected SC in the thoracic segment and found that muscle atrophy in the paralyzed hindlimbs was improved, which was associated with increased survival of motor neurons and NT-3 expression in the lumbar SC. 71 This may be attributed to the transmission of electrical stimulation to the SC segment through primary afferent nerve fibers. 40,66,72 NT-3 also plays an important role in neuroprotection, axon regeneration, and synaptic plasticity. 73,74 The effects of GV-EA on the survival of motor neurons in the lumbar SC are speculated to be related to an increase in NT-3 levels. 71 GV-EA also stimulates the release of neurotransmitters (such as CGRP and acetylcholine, among others) in the lumbar SC, which enhances the excitability of specific neuronal circuits that lead to the contraction of target muscles innervated by the surviving motor neurons, thereby alleviating muscle atrophy. 40

| GV-E AALLE VIATE SSCIBY PROMOTINGTHEINTEG R ATI ONOF TR AN S PL ANTEDS TEMCELL S
Approximately half of all SCIs involve complete SC transection, with little or no remaining nerve tissue in the injured segment. Restoration of SC tissue-for example, through cell transplantation-is essential for the effective treatment of SCI. 1,75 NSCs or MSCs transplanted into the injured SC are induced to differentiate into neurons or neuron-like cells and glial cells that restore SC structure and function. Transplanted NSCs have been shown to differentiate into neurons and glia, thereby reducing secondary damage to the SC and improving spinal motor function. 76 However, they are also more likely to differentiate into astrocytes than neurons, which is a major barrier to the clinical application of cell-based therapies for SCI.
One way to overcome this problem is to use GV-EA in combination with NSC transplantation to treat SCI. 77  However, it is important to consider that there are key ethical barriers to NSC transplantation that limit its translational potential.
In clinical studies of spinal cord treatment, the embryonic neural stem cells (NSCs) that are used are generally derived from human embryonic brain tissue or human embryonic spinal cord tissue. The ethical debate over whether embryonic NSC technology is "lifedamaging" was triggered by the destruction of embryos caused by selective abortion. 81 However, most researchers agree that the process of consent for human embryonic neural stem cell research, and its clinical application, is fully informed and voluntary. 82 Due to the fact that this is still an area associated with significant debate, the application of neural stem cells may be limited to translational clinical research for the time being. In contrast, there is no risk of immunological rejection or ethical issues associated with the transplantation of autologous MSCs; this process has been used successfully for the repair of bone, cartilage, tendon, and muscle tissues. 83 Thus, MSC transplantation may also be used to repair injured SC. 84,85 A previous in vitro study demonstrated that NT-3 induces MSCs to overexpress the NT-3 receptor (TrkC) to differentiate into neuron-like cells. 48,86 Within the last 10 years, studies in our laboratory, and others, demonstrated that the combination of EA and MSC transplantation provides a greater advantage than either procedure alone when applied to animals with SCI. 77,[87][88][89][90] Based on this observation, we used a combination of GV-EA and the transplantation of TrkC gene-modified MSCs to treat SCI. 48,87,91,92

Regenerating nerve fibers on in the injury/ graft site
EA+grafted NSCs [77] Rat/Adult/Female Rat hippocampus Showing neuron-like cells and astrocyte-like cells

No nerve fiber regeneration shown
EA+grafted MSCs [87] Rat/Adult/Female Rat hippocampus Showing neuron-like cells and oligodendrocyte-like cells

Showing nerve fiber regeneration
EA+grafted BMSCs [88] Rat/Adult/Male Rat bone marrow Showing neuron-like cells and astrocyte-like cells No nerve fiber regeneration shown EA+grafted MSCs [92] Rat/Adult/Female Rat bone marrow No cell differentiation was shown Showing nerve fiber regeneration EA+grafted BMSCs [89] Rat/Adult/Male Rat bone marrow No cell differentiation was shown No nerve fiber regeneration shown EA+grafted MSCs [91] Rat

| GV-E AS TIMUL ATE STHEREPAIRBY TR ANS PL ANTEDMSC SOFDEMYELINATION INJ URYINTHESC
Both mechanical and chemical SCI can lead to nerve fiber demyelination. 98 We have previously shown that GV-EA promotes the synthesis and secretion of NT-3 in demyelinated areas of the SC and increases the number of endogenous oligodendrocyte precursor cells. 50,99 However, the number of such cells is limited, and even MSCs transplanted into the injury site rarely differentiate into oligodendrocyte-like cells. 87   of clinical settings. In addition, some researches indicated that sex differences exist a certain effect on the repair of central nervous system injury. 10 8-114 Hence, future study should pay attention to the potential influence of sex differences on electro-acupuncture and its combination with adult stem cell transplantation for spinal cord injury treatment.

ACK N OWLED G M ENTS
This research was supported by grants from National Natural Science Foundation of China (No. 81891003; 81674064; 81774397) to Y.S. Zeng and Y. Ding.

CO N FLI C TO FI NTE R E S T
The authors declare no conflict of interest.

DATAAVA I L A B I L I T YS TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.