Modulation of neurogenesis via neurotrophic factors in acupuncture treatments for neurological diseases

Accepted Manuscript Review Modulation of neurogenesis via neurotrophic factors in acupuncture treatments for neurological diseases Hwa Kyoung Shin, Sae-Won Lee, Byung Tae Choi PII: DOI: Reference:

S0006-2952(17)30246-0 http://dx.doi.org/10.1016/j.bcp.2017.04.029 BCP 12808

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

9 February 2017 26 April 2017

Please cite this article as: H.K. Shin, S-W. Lee, B.T. Choi, Modulation of neurogenesis via neurotrophic factors in acupuncture treatments for neurological diseases, Biochemical Pharmacology (2017), doi: http://dx.doi.org/ 10.1016/j.bcp.2017.04.029

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Modulation of neurogenesis via neurotrophic factors in acupuncture treatments for neurological diseases

Hwa Kyoung Shina,b,c,d, Sae-Won Leea,b, Byung Tae Choia,b,c,d*

a

Department of Korean Medical Science, School of Korean Medicine, Pusan National

University, Yangsan 50612, Korea b

Graduate Training Program of Korean Medicine for Healthy-aging, School of Korean

Medicine, Pusan National University, Yangsan 50612, Korea c

Korean Medical Science Research Center for Healthy Aging, Pusan National University,

Yangsan 50612, Korea d

Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National

University, Yangsan 50612, Korea

* Corresponding author: Professor Byung Tae Choi, Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan 50612, Republic of Korea Telephone: +82-51-510-8475; Fax: +82-51-510-8437; E-mail: [email protected]

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Abstract Acupuncture is one of the main healing arts in Oriental medicine. It has long been used in East Asian countries, including Korea and China, and is thought to be an effective alternative treatment for various neurological diseases. The therapeutic effects of acupuncture come from inserting a needle at specific acupoints on the body surface, with subsequent delivery of stimulation via manual rotation or electric pulses (electroacupuncture, EA). In various neurological disease models, peripheral nerve stimulation using acupuncture or EA may have protective effects on neural tissues by increasing expression of neurotrophic factors (NTFs), such as brain-derived neurotrophic factor and glial-derived neurotrophic factor, in the central nervous system, especially the brain. In addition, acupuncture may contribute to recovery from functional impairments following brain damage by encouraging neural stem cell proliferation, which is active at the initial stage of injury, and by further facilitating differentiation. Hence, acupuncture may act as a stimulator activating peripheral nerves at specific acupoints and inducing the expression of various NTFs in the brain. Subsequently, NTFs induced by this treatment trigger autocrine or paracrine signaling, which stimulates adult neurogenesis, thereby exerting therapeutic effects on functional impairments in neurological diseases. Acupuncture may offer an alternative treatment that promotes adult neurogenesis through the expression of NTFs in the brain. It may also have synergistic effects when combined with pharmacological interventions, again facilitating neurogenesis. This review examines recent studies concerning the effects of acupuncture and EA on adult neurogenesis associated with NTF expression in neurological diseases, in particular stroke, Alzheimer’s disease, and Parkinson’s disease.

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Keywords: Acupuncture, Neurotrophic factor, Neurogenesis, Stroke, Alzheimer’s disease, Parkinson’s disease

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1. Introduction

Acupuncture, a major medical tool in East Asia with a long history, has been used in the treatment of numerous diseases. Acupuncture is an economical treatment without adverse effects and so has been widely used for the treatment of various neurological diseases, as well as being used in post-illness rehabilitation to aid recovery from functional impairments [1-5]. Traditional explanations for the therapeutic effects of acupuncture are based on the meridian system [6], but such traditional hypotheses are controversial and are difficult to examine empirically. Thus, the mechanisms underlying the effects of acupuncture are not fully understood. Nonetheless, acupuncture is extensively applied in today’s aging society as an alternative or complementary treatment for neurological impairments, including sensorimotor dysfunction, cognitive impairments, and depression, without any obvious side effects [1, 3-5, 7]. It is well known that neurogenesis, the continued growth and development of neurons, can occur throughout life in specific regions of the adult brain, such as the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus [8, 9]. Neurological disorders that cause brain damage activate endogenous neural repair systems, such as neurogenesis. Therefore, modulation of this activation and mechanisms underlying such endogenous recovery systems offer attractive therapeutic potential for the treatment of neurological disorders [9-11]. A remarkable insight into adult neurogenesis was the discovery that the persistence and regulation of neurogenesis is readily influenced by extrinsic signals involving neurotrophic factors (NTFs) [9, 12]. Moreover, various NTFs have common roles in both embryonic and adult neurogenesis [9]. Functional recovery from impairments caused by neurological diseases requires proliferation, survival, differentiation, and migration of neural 4

progenitor/stem cells (NPCs/NSCs), which temporarily increase following brain damage, as well as eventual functional integration with the central nervous system (CNS) [11, 13]. In animal models of neurological diseases such as stroke, Alzheimer’s disease (AD), and Parkinson’s disease (PD), acupuncture has been shown to have neuroprotective effects and to enhance functional recovery by activating and increasing expression of brain NTFs, including brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) [1417]. Acupuncture exerts its therapeutic effects by activating and facilitating adult neurogenesis via the stimulation of NSC proliferation and differentiation in the brain [15, 18, 19]. Therefore, one possible neurophysiological mechanism underlying the therapeutic effects of acupuncture is the regulation of plasticity in the brain (e.g., neurogenesis). NTF levels, which are modulated by the therapeutic effects of acupuncture, are associated with enhancement of survival, proliferation, and differentiation of NSCs [17, 18, 20]. Better understanding of endogenous neurogenesis could provide opportunities to develop novel strategies in the treatment of neurological diseases. Crucially, acupuncture could also be used in combination with drugs or other therapeutic treatments to stimulate neurogenesis in these diseases. Many previous studies have been conducted to investigate treatment of neurological diseases by promoting neurogenesis, including studies of complementary, alternative, and integrative medicine [4]. The results from these studies have suggested that acupuncture enhances the expression of NTFs in the brain, and that these NTFs may stimulate neurogenesis, thereby exerting a therapeutic effect on functional recovery in neurological diseases [15, 18, 19]. In this review, we report on recent trends and studies of the therapeutic effects of acupuncture in neurological diseases, especially in stroke, AD, and PD, which share symptoms such as cognitive impairment and depression, focusing on studies of adult neurogenesis and NTF expression in the brain.

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2. Properties of acupuncture as peripheral nerve stimulation

According to traditional Oriental medical theory, the meridian system is widely distributed inside and outside the body, including the skin and internal organs. It forms a network, serving an integrative role and creates functional associations within the entire body [21]. Thus, lesions of the internal organs are exposed to the surface of the body through the meridian system, and external stimuli from a treatment are delivered to the internal organs. If the meridian system is described using the concept of a line, the meridian point (acupoint) is described using the concept of a dot distributed as major spots above the meridian system. The acupoint refers to the empty space above the meridian system, which can collect qi (vital substances) and xue (blood) as a healthy physiological process, or morbid energy as part of pathological processes. Therefore, diseases are diagnosed by examining pain and color changes at these acupoints, and therapeutic effects are obtained by adjusting the condition of the internal organs via stimulation using needles. In other words, the acupoint is not only a diagnostic site but also a therapeutic spot, acting as a door that connects the meridian system to the external space [6, 21]. For acupuncture treatment, the proper acupoint is selected according to each different disease, and a needle is inserted [6, 22]. To maximize the therapeutic effect, hand manipulation, such as spinning or flicking the needle, is performed to trigger proper stimulation [22]. Although clear evidence of an acupuncture-specific response in the brain and spinal cord remains elusive, stimulation of the acupoint using needles activates the CNS, i.e., the spinal cord and brain, via afferent nerve pathways of the peripheral nervous system and induces various neurophysiological changes [20, 23-25]. Furthermore, a model has been proposed in which stimulation from the needles passes through the CNS and acts on the internal organs via brain-gut axes, such as the hypothalamus-pituitary-axis [26]. In summary, 6

a framework in which acupuncture activates the brain and spinal cord is essential, and has been accepted in studies of the therapeutic mechanisms of the treatment [23, 24]. In addition to the selection of a specific acupoint, the method of stimulation is also crucial in acupuncture. Today, electric pulses, magnetic fields, ultrasound, and lasers that are used in other physical therapies can also be applied in acupuncture instead of hand manipulation after traditional needle insertion [27, 28]. In particular, electroacupuncture (EA), which creates electrical stimulation, allows the modulation of constant pulse width, intensity, and frequency, making it easier to standardize treatment than in traditional manual acupuncture, and aids elucidation of underlying mechanisms [20]. Hence, EA is widely used in the experimental and clinical fields. Needle insertion at acupoints followed by manual rotation or electric stimulation activates all types of afferent fibers [20, 23]. These impulses ascend mainly via the ventrolateral funiculus, and subsequently involve many brain nuclei creating a complicated network that processes therapeutic effects [24]. Therefore, it is proposed that the therapeutic efficacy of acupuncture is based on the stimulation of sensory nerve fibers as a potent form of somatosensory stimulation, and the subsequent activation of physiological processes in the CNS [20, 25]. Although there is a lack of novel evidence for specific CNS responses to acupuncture, neurotransmitters, neuropeptides, or cytokines have been proposed as possible mediators of the therapeutic actions of acupuncture [20, 29, 30]. Moreover, acupuncture stimulation induces very similar activation of physiological processes to those resulting from physical stimulation [20, 31]. Adult neurogenesis is also responsive to environmental changes. Electric stimulation and aerobic physical exercise cause the release of NTFs and promote angiogenesis, thereby facilitating neurogenesis and synaptogenesis [32, 33]. Activity-associated metabolic stresses, such as physical exercise, cognitive stimulation, and dietary restriction, result in the production of proteins involved in 7

neurogenesis leading to neuronal survival [9, 34]. Such activity-associated therapies could be applied to promote regeneration in injured nerves and prevention for neurological diseases [32, 35]. Therefore, both traditional acupuncture and the various kinds of acupuncture involving EA act as extrinsic signals for neurogenesis by stimulating specific points on the body surface, thereby increasing the physiological activity of the CNS [20, 23, 24], especially enhanced activity of NTFs in the brain [15, 18, 19]. In other words, increased activity of NTFs following acupuncture or EA is highly likely to exert therapeutic effects via neurophysiological changes, and therefore have similarities with activity-associated therapies such as exercise, cognitive stimulation, and dietary restriction [20].

3. Activation of neurotrophic factors in the central nervous system by acupuncture

Several studies have investigated NTF changes in the CNS using acupuncture in neurological diseases. A PubMed database search for articles regarding acupuncture or EA associated with NTFs, written in English, revealed that the majority of reports have investigated animal models and there are few clinical studies on humans. Studies using animal models of neurological diseases include studies on stroke, AD, PD or cognitive impairment caused by ischemia, and depression. In stroke animal models, which are the most widely reported, the stimulation of highly used acupoints, such as Baihui (GV20) [18, 36-42], Dazhui (GV14) [18, 37, 39, 42], and Quchi (LI11) [15, 42, 43] led to improved neurobehavioral function damaged by ischemia. These beneficial effects are mainly caused by enhanced expression of various NTFs, including BDNF [15, 18, 36-39, 41, 43], GDNF [41-43], vascular endothelial growth factor (VEGF) [18], and stromal cell derived factor-1α (SDF-1α) [37]. In general, these studies 8

reported beneficial effects, with the alleviation of neuronal death. However, in some studies cell proliferation was the focus [15, 18, 43], while in others differentiation of neuronal progenitor cells was investigated [18]. However, there are also reports showing no significant change in the infarct volume or BDNF levels despite motor function recovery [44]. In PD models, studies have reported on the stimulation of highly used acupoints, Baihui and Dazhui [14, 45-47], which results in the activation of BDNF [45, 47, 48] and GDNF [14, 48]. Furthermore, such stimulation also leads to protection against degeneration of dopaminergic neurons [14, 45, 46] and alleviation of depression [47]. In cognitive impairment models either accompanied by AD or not, the acupoint Baihui [49-52] was stimulated in most cases, resulting in the increased expression of BDNF [17, 49-52] and improved cognitive function [17, 49-52]. Moreover, the stimulation of Baihui and Yintang (Ex-HN3) [16, 53], two highly used acupoints, results in the activation of BDNF and leads to amelioration of depression-like behaviors and dysfunction [16, 53, 54]. Among the acupoints that were used in these articles using animal models, the use of Baihui was the highest, followed by Dazhui, and Quchi. In addition, Zusanli (ST36) has also been widely used in neurological disease [15, 43, 54]. Identified NTFs included BDNF and GDNF, indicating that reports on BDNF and GDNF accounted for the majority. The acupoint Baihui (located on the head) and Dazhui (on the neck) belong to the same Governor vessel, i.e., the main portion of the meridian that ascends along the midline of the back to the head and then descends along the midline of the face [55]. Baihui includes branches of the trigeminal and second cervical nerve and Dazhui includes the eighth cervical nerve [56]. The Zhusanli and Quchi are located on the leg and arm, and belong to the Stomach and Large Intestine Meridian, respectively. Zhusanli includes branches of the deep peroneal and tibial nerve and Quchi includes the radial nerve [56]. Previous studies report that the stimulation of specific acupoints, stimulating different nerve branches, exerts therapeutic effects in different 9

diseases such as stroke, AD, and PD by the common mechanism of increasing expression of NTFs, such as BDNF and GDNF, in the brain [14, 15, 17, 18, 45, 47, 51, 52]. When EA stimulation is applied in the bilateral common carotid artery stenosis (BCAS) model, the expression of various growth factor genes involving Figf (c-fos induced growth factor), Mdk (midkine), and NT4/5 (neurotrophin-4/5) markedly increases in the corpus callosum in the brain [19]. Moreover, NTFs were expressed in numerous brain regions after EA stimulation. Increased expression of BDNF following EA was observed throughout the whole brain in ischemic stroke models, with expression observed in diverse regions including the cerebral cortex, striatum, and hippocampus, rather than in one specific region [15, 18, 50]. These results may suggest that EA functions as a stimulator or enhancer of NTF activity throughout several brain regions. NTFs increased by acupuncture, such as BDNF and GDNF, have several well-known functions. BDNF is a neurotrophin that plays a critical role in neuronal survival and synaptic plasticity, and is also an important modulator of developmental events in the nervous system, including proliferation and differentiation [34, 57, 58]. Although adult neural progenitors are regulated by a variety of other extrinsic factors, BDNF and its receptor tyrosine receptor kinase (Trk) B are key positive regulators of adult neurogenesis [9]. GDNF was first identified as a survival factor for dopaminergic neurons of the midbrain [59]. The GDNFfamily of ligands comprises GDNF and related growth factors (neurturin, artemin, and persephin), which all belong to the TGFβ family [60]. GDNF, along with the GDNF-family ligands plays a crucial role in the development and maintenance of central and peripheral neurons [60]. GDNF is involved in the survival and differentiation of several peripheral neurons [60]. Furthermore, GDNF has been identified as a ligand promoting neurogenic differentiation of NPCs [61]. The underlying mechanisms by which NTFs induce neuroprotection and neurogenesis are 10

not fully understood. NTFs induced by acupuncture may be involved in mechanisms underlying proliferative and differentiation of NSCs, restoring injured neurons in neurological diseases [62]. Thus, acupuncture acting as a potential stimulator of endogenous responses via NTFs provides an attractive therapeutic target for neurological diseases.

4. Modulation of neurogenesis by acupuncture in neurological diseases

Table 1 provides a summary of studies that have investigated the effects of acupuncture on neurogenesis in stroke, AD, and PD, where there are similar symptoms.

4.1. Effect of acupuncture on the neurogenesis in stroke

Acupuncture has long been widely used in patients with stroke or during rehabilitation after stroke. The recovery of functional impairments via acupuncture after stroke has been actively studied, with recent studies suggesting that acupuncture promotes neurogenesis and is therefore a promising treatment for the recovery of neuronal dysfunction due to stroke [3]. In particular, acupuncture has been shown to effect neurogenesis from day 7 to day 14 after experimental ischemic stroke, suggesting an optimum time window in stroke for acupuncture therapy [3]. Studies of the treatment of stroke using acupuncture have reported reduced cerebral ischemic injury and facilitation of recovery of neuronal deficits after stroke [18, 19, 63]. The most used acupoints in experimental stroke models are Zusanli [15, 43, 64-68] and Quchi [15, 43, 65-68] and Baihui [18, 19, 69, 70] either individually or in combination. Nonetheless, Baihui is the principle acupoint that is often selected for stroke patients [3]. Acupuncture treatment for neuronal deficits caused by focal ischemia injury elicits multiple interacting mechanisms, involving improved cerebral blood supply, regulation of 11

gene transcription, and neurogenesis [63]. Acupuncture increases cell proliferation in the dentate gyrus of gerbils following ischemic injury [64]. Furthermore, EA treatment promotes NSC proliferation and neurogenesis in the dentate gyrus area via upregulation of Notch signaling and promotion of cell cycle progression [43, 69]. EA significantly alleviates cerebral ischemic injury by promoting proliferation of endogenous NSCs in the inferior zone or SVZ of the lateral ventricle, and stimulates differentiation of proliferating NSCs into astrocytes and maturation into neurons [65, 71]. EA treatment after ischemic stroke promotes post-stroke recovery of behavioral function by enhancing the proliferation and differentiation of NSCs into neurons or astrocytes via BDNF and VEGF signaling pathways [18]. EA stimulates the proliferation and self-renewal of NPCs in the cortical peri-infarct area after stroke by upregulating Wnt/β-catenin signaling [66]. EA treatment, which activates both the extracellular signal-regulated kinase (ERK) pathway and the expression of cell cycle-regulatory proteins promoting neural cell proliferation, plays a protective role in the brain, and thereby improves neurological function [67, 68]. Some of these studies focus on the levels of NTFs as one possible biological mechanism by which neurogenesis is enhanced by acupuncture. EA activates the secretion of BDNF and GDNF [43], and the expression of BDNF and VEGF is also increased in the SVZ and hippocampus of ipsilateral side [18]. Combined treatment with EA and NGF shows a synergistic effect on proliferation and survival of progenitors after focal cerebral ischemia, and this contributes to improved neurological function and reduced infarct volume [70]. EA promotes the proliferation of reactive astrocytes by regulating cell cycle phase transitions and enhancing local expression of BDNF in the peri-infract cortex and striatum [15]. Although the SVZ and SGZ are known as major regions of adult neurogenesis, severe brain injuries resulting from stroke can elicit a latent neurogenic program in non-neurogenic brain regions, including the striatum [72, 73]. The presence of these reactive striatal astrocyte12

derived neurons, originating from local astrocytes rather than from major neurogenic regions, provides new therapeutic strategies for enhancing brain repair [15, 72, 74, 75]. Moreover, EA stimulation promotes the regeneration of oligodendrocytes by the process of oligodendrocyte differentiation from parenchymal oligodendrocyte precursor cells via NT4/5-TrkB signaling [19]. Therefore, in addition to typical adult neurogenesis, during which neurons are generated from NSCs, the growth and differentiation of neurons through latent neurogenic programs, trans-differentiation of astrocytes caused by brain damage or parenchymal oligogenesis is an emerging therapeutic strategy for the treatment of neurological diseases.

4.2. Effect of acupuncture on neurogenesis in Alzheimer’s disease and cognitive impairments

AD is a severe progressive neurodegenerative disease, for which the major clinical symptoms include dementia, memory loss, personality disorder, and language problems [76]. Because the link between pathological phenomena in AD and acupuncture has not been clearly elucidated, there are fewer studies of neuronal function recovery involving neurogenesis following acupuncture in AD than in stroke. AD is caused by various factors and there is currently no effective treatment. However, acupuncture has been shown to have beneficial effects to a certain extent as an alternative and easy-to-perform treatment [4, 50]. Therefore, some studies have examined the therapeutic effects of acupuncture on cognitive impairment that accompanies AD, and the related signaling associated with NTF expression [50]. Acupuncture induces cell proliferation along the dorsum of the alveus hippocampi, extending from the lateral ventricle to the corpus callosum, and this is associated with improvement of cognitive deficits in senescence-accelerated mouse prone 8 mice (SAMP8), which shows similar neuropathologic characteristics to AD [77]. EA improves cognitive 13

functions such as the learning and memory in AD mice (amyloid precursor protein (APP)/presenilin 1 (PS1) double transgenic mice) and promotes neurogenesis in the cortex and hippocampus associated with decreased Aβ deposits and increased BDNF expression [50]. Thus, EA affects BDNF and its downstream pathway induces neurogenesis, improving neurobehavioral impairments in the AD model [50]. EA can also effectively alleviate cognitive dysfunction following brain irradiation, but these effects involve synaptophysin and are not dependent on hippocampal neurogenesis [78]. BDNF plays crucial roles in the long-term survival of newborn neurons [79], but Aβ deposits in AD disturb BDNF-activated pathways, such as Ras/ERK, phosphatidylinositol 3kinase (PI3K)/Akt, and protein kinase A (PKA)/cAMP response element-binding protein (CREB) [80, 81]. Previous studies of acupuncture for AD report reductions in Aβ deposits and beneficial effects on cognitive dysfunction, with a focus on the stimulation of neurogenesis and increased BDNF expression in the brain [50].

4.3. Effect of acupuncture on neurogenesis in Parkinson’s disease and depression

A major pathological characteristic of PD is motor symptoms caused by dopaminergic neuron loss in the substantia nigra pars compacta [82]. Treatment by encouraging proliferation and supplementation of dopaminergic neurons via neurogenesis is a very attractive option. However, it is controversial whether neurogenesis occurs in the substantia nigra, as well as whether migration from known neurogenic areas can occur [83, 84]. Acupuncture has been shown to have effects in PD as an adjunct to existing treatments, and the prospect of acupuncture as a potential alternative therapy for PD is promising [2, 7]. However, there have been no studies of neurofunctional recovery associated with increased neurogenesis in PD mediated by acupuncture, and most studies investigate NTFs, such as 14

BDNF and GDNF and their downstream signaling [14, 45, 47, 48]. However, the non-motor symptoms associated with PD such as hyposmia, anhedonia, depression, and anxiety occur nearly a decade or more before the first signs of motor symptoms, and are not directly related to the neurodegenerative loss of dopaminergic neuron in the substantia nigra [85-87]. Studies of the effects of acupuncture on models of depression, one of the major non-motor symptoms of PD, suggest that EA generates a clear antidepressant effect on the chronic unpredictable stress-induced depression model by promoting hippocampal progenitor cell proliferation [87-89]. EA is also associated with ERK activation leading to cell proliferation and preservation of quiescent neural progenitors from apoptosis [89]. It also interferes with the hippocampal microenvironment and enhances the activation of ERK signaling pathways, mediating the beneficial effects of EA on NSC proliferation [88]. Moreover, numerous genes associated with the onset of PD are also related to embryonic or adult neurogenesis, and some studies report that damaged adult neurogenesis contributes to the non-motor symptoms of PD [85, 86]. Thus, the therapeutic potential of acupuncture for non-motor symptoms of PD such as depression via increased NTF activity cannot be excluded.

5. Possible neurogenic mechanisms of neurotrophic factors following acupuncture

The point of interest in regards to such studies is how somatosensory (mechanical) stimulation such as acupuncture might regulate NTF expression. As similar physiological processes result from activity-associated therapies, the activation of NTF signaling by acupuncture may involve the major excitatory neurotransmitter glutamate and its receptor [20, 25, 32, 35]. EA and glutamate receptor antagonists show synergistic anti-nociceptive effects in an inflammatory pain model, suggesting an interaction between EA and glutamate 15

receptors [90, 91]. EA treatment has generally been shown to have a therapeutic effect via attenuation of high expression of ionotropic glutamate receptors or by suppressing Ca2+ influx [92-95]. In addition to these effects in disease, EA has been shown to induce analgesia via peripheral electrical stimulation in healthy animals by enhancing the activation of glutamate receptors [96-98]. These studies may support the proposal that acupuncture activates dormant therapeutic neuronal networks that evoke the release of NTFs via excitatory neurotransmitters, such as glutamate, in competition with pathophysiological networks, exerting eventual therapeutic effects [23, 99]. Acupuncture may enhance the release of the excitatory neurotransmitter glutamate and thus increase activation of its receptor [96-98]. Activation of glutamate receptors, such as Nmethyl-D-aspartate receptors, promotes influx of Ca2+ and activates kinases, including Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) [34, 100]. These kinases are known to stimulate numerous transcription factors, such as CREB [101, 102], nuclear factor-kB (NF-kB) [103, 104], activator protein-1 (AP-1) [105], and betacatenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) [106]. It was reported that BDNF was induced at the transcriptional level by CREB [34, 107, 108] and NF-kB [108, 109]. Furthermore, in terms of GDNF gene regulation, the GDNF promoter contains binding sites for transcription factors, including CREB and NF-kB [110]. The transcription of GDNF has been shown to be stimulated by CREB phosphorylation and c-Fos activation, a component of the AP-1 transcription factor [111, 112]. CREB activation is usually accompanied by enhanced BDNF expression following acupuncture stimulation [16, 19, 49, 52, 53]. Therefore, acupuncture treatment may induce the production of growth factors, such as BDNF and GDNF, in neurons and glial cell via the excitatory transmitter glutamate in the same way as activity-dependent therapy [113] (Fig. 1). In previous studies of acupuncture, the downstream pathways activated by NTFs and 16

transcription factor activation have been investigated. In a stroke model, the activity of the GDNF family receptor RET increases upon EA treatment along with the activation of downstream PI3K/Akt signaling, exerting protective effects on neurons [42, 114]. Activity at this receptor can also induce BDNF-mediated neuroprotection against neuronal apoptosis via Raf/ERK signaling [39]. Acupuncture and EA have also been shown to improve cognitive function by increasing BDNF levels, as well as increasing activity along the CREB or PKA/CREB signaling pathways, in cognitive impairment models [49, 52]. In a depression model, EA stimulation had an alleviating effect on depression by increasing BDNF levels, as well as ERK/CREB or TrkB/PKA/CREB signaling [16, 54]. Meanwhile, in the hippocampus of telomerase-deficient mice, EA induces BDNF upregulation and increases activity of several downstream proteins including TrkB, p75NTR, Akt, and ERK1/2 [115]. Furthermore, EA also increases BDNF and CREB activity in control animals [116]. In summary, studies of the effects of acupuncture on neuronal survival and protection in the adult brain suggest that it induces NTFs (mainly BDNF and GDNF), which may enhance proliferation of NSCs or induction of neuronal differentiation via downstream signaling [9, 60]. BDNF-mediated signaling through TrkB is important for adaptive responses, mediating progenitor cell survival and neurogenesis via regulation of various downstream signals, such as MAPK and PI3K, which regulate cell survival and generation, respectively [57, 117-120]. Whereas, GDNF induces Ret-dependent signaling, which switches on signaling pathways involved in the proliferation and differentiation of neurons via Ras/MAPK and PI3K/Akt signaling [121-123]. Moreover, the transcription factor CREB plays essential roles in BDNF transcription and its activation is associated with progenitor cell mitosis [9, 124]. However, unlike reports of the effects of EA on neuroprotection, there are few studies of NSC proliferation facilitated by acupuncture associated with NTF downstream signaling. In an MCAO model, Wnt/β-catenin signaling was shown to be involved in the NPC proliferation 17

induced by EA [66], and increased BDNF and GDNF levels, in addition to the activation of Notch signaling, appear to facilitate NSC proliferation and neurogenesis [43, 69]. EA activates the ERK signaling pathway and stimulates cerebral cell proliferation, exerting a neuroprotective effect via ERK and a microenvironment for NSC proliferation [67, 68, 88]. EA enhances proliferation and differentiation of NSCs via the BDNF or VEGF/PI3K signaling pathway [18]. BDNF and NT4/5 also have very similar targets. EA stimulation promoted the recovery of memory function in a vascular dementia model via a mechanism that promotes gliogenesis and involves NT4/5-TrkB signaling [19]. These studies suggest the involvement of Wnt/β-catenin and Notch signaling, which play important roles in NSC proliferation and differentiation [125, 126], and support a mechanism by which acupuncture increases the expression of NTFs, such as BDNF and GDNF [43]. However, most studies have discussed the importance of PI3K/Akt and Raf/ERK cell signaling pathways mediated by the Trk receptor, which induces the transcription factor CREB and mediates neuronal function via neuronal proliferation and survival [18, 19, 67, 68, 88]. Owing to the small number of fragmentary studies conducted, there have been no reports presenting direct evidence of functional recovery using acupuncture resulting from activation of neurogenesis, including NSC/NPC proliferation, differentiation, and functional integration with the CNS. In addition, if acupuncture mainly increases expression of BDNF and GDNF, this neurotrophin and the GDNF family of ligands could subsequently activate common intracellular downstream phosphorylation signaling via their receptors [60]. In other words, the expression of BDNF and GDNF induced by acupuncture is likely to exert therapeutic effects, not by independent NTF receptor downstream signaling, but by the expression of different kinds of NTFs and the activation of receptors sharing common intracellular signaling, which ultimately converge on common transcription factors, such as CREB [9]. 18

Therefore, acupuncture may act as a stimulator or an enhancer of the production of NTFs, which will act as autocrine or paracrine signals to promote the NSC/NPC proliferation and differentiation to neuroblasts, as well as promoting cell survival. This could lead to functional integration of new neurons into the CNS, thereby resulting in functional recovery (Fig. 2). Furthermore, the function of NTFs for which acupuncture increases their expression could be inferred as signal transducers stimulating dormant NSCs to enter neurogenesis, as shown in young and healthy models of neurogenesis [116, 127].

6. Conclusion and future perspectives

The idea that adult neurogenesis can achieve integration within the CNS and thereby induce functional recovery is very attractive [9, 10]. Although the association between adult neurogenesis and recovery of neurological impairments remains ambiguous, studies showing increased expression of NTFs in the brain following acupuncture treatment are noteworthy. If acupuncture could be used as a treatment to increase NTF expression as an extrinsic signal for neurogenesis, it might offer promise as an additional treatment in a variety of disorders. Hence, in the following sections we consider some key points gleaned from studies of the correlation between NTF expression induced by acupuncture and the promotion of neurogenesis. We also consider the clinical implications of these findings and how our conclusions might extend the application of acupuncture in neurological diseases.

6.1. Use of acupuncture as a stimulator for neurotrophic factors in the brain

In addition to many studies showing that acupuncture increases expression of BDNF and GDNF in the brain, in various models of neurological diseases, there are also studies 19

showing that acupuncture increases expression of other NTFs, such as NGF, NT3, NT4/5, ciliary neurotrophic factor (CNTF), and SDF-1α [19, 37, 70, 128]. Despite the limitations of these studies, acupuncture stimulating specific acupoints on the body surface could potentially induce the expression of various NTFs in different regions in the brain [15, 18, 50]. Although increased expression of one specific kind of NTF following acupuncture could exert a therapeutic effect, it is also possible that different kinds of NTF work concordantly to provide clinical benefits. The aforementioned NTFs, BDNF and GDNF, may act on neurogenesis via common intracellular signaling after the activation of different receptors [61, 129]. Therefore, it is essential to conduct further study of acupuncture as a stimulator of various NTFs, by which neurogenesis may be increased in neurological diseases.

6.2. Therapeutic effects of stimulation at the same acupoint in different neurological diseases

In studies of NTF expression following acupuncture, specific acupoints (Baibui, Dazhui, and Quchi) were widely used, although they were used in different neurological diseases models. The therapeutic principle of acupuncture according to traditional Oriental medicine is based on the meridian system, which refers to 14 lines connecting the 360 known acupoints [6]. This hypothesis acknowledges the specificity of acupoints, describing the function of each acupoint for particular diseases, and different combinations of multiple acupoints are used in patients with neurological diseases [6, 22]. If acupuncture treatment is viewed as a stimulator of NTFs and thereby promotes neurogenesis, studies of specific acupoints that effectively induce the activation of NTFs such as BDNF and GDNF in the brain may be more significant than the selection of acupoints for different diseases according to the traditional hypothesis.

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6.3. Mechanistic studies of acupuncture regarding stimulation of specific acupoints

The mechanism underlying acupuncture’s therapeutic effects has been proposed to be similar to that of activity-associated therapy, including exercise, enriched environment, stimulation, and dietary energy restriction, in which mild stresses are applied to neuronal tissues [20, 33, 34]. However, unlike these, acupuncture exerts therapeutic effects by stimulating the peripheral nervous system at specific acupoints on the body surface. That is, physical stimulation from a needle of peripheral nerves of a specific area induces neurophysiological changes in the CNS [20]. Therefore, in addition to selecting specific acupoints that promote the expression of NTFs, elucidating the therapeutic targets in specific brain networks in which neurophysiological activity of neurotransmitters or neuromodulators is modulated by specific acupoint stimulation might be critical for developing a more efficient acupuncture therapy.

6.4. Acupuncture treatment as a non-pharmacological intervention or combination therapy Neurological diseases accompanying brain tissue damage induce proliferation of neurons by triggering active neurogenesis at the initial stage of disease onset as a defense mechanism [3, 9, 10]. However, most of these neurons die within 1–2 weeks without being functionally integrated into the CNS [130]. Functional recovery in neurological diseases via neurogenesis, as well as their functional integration into the CNS, is a desirable treatment. Therefore, acupuncture, which can achieve this, could be an alternative treatment. As an economical medical technology without adverse effects, acupuncture could be used along with other treatments for neurological diseases, and could be used in patients who cannot undergo drug or exercise therapy [34]. Furthermore, using acupuncture along with other therapies to promote neurogenesis, especially pharmacological interventions that increase 21

NTF production or activate their receptors, might help adjust the dose of drug administration and create synergistic treatment effects.

Conflicts of interest

All authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2014R1A5A2009936). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (2015R1A2A2A03006712).

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Legends for Figures

Fig. 1. Schematic diagram of the proposed mechanism of NTF expression induced by acupuncture stimulation. Somatosensory (mechanical) stimulation, such as acupuncture and electroacupuncture, promotes glutamate release, which increases activation of the glutamate receptor. In turn, this results in Ca2+ influx, thereby activating CaMKII and PCK. The promoter regions of BDNF and GDNF contain several transcription factor binding sites for CREB, NFkB, TCF/LEF, and AP-1. It has been reported that acupuncture stimulates transcription of BDNF through CREB (solid arrow). NFkB, TCF/LEF, and AP-1 could potentially regulate BDNF or GDNF transcription because these transcription factors are regulated by Ca2+ influx, and their binding sites are present in the BDNF and GDNF promoter region (dashed arrow).

Fig. 2. Schematic diagram of the proposed mechanism of neurogenesis induced by acupuncture. Needle insertion to acupoints activates afferent nerve fibers of the spinal or cranial nerve, and upregulates NTFs in the brain via physiological processes involving the excitatory neurotransmitter glutamate in the central nervous system. Secreted NTFs, such as BDNF and GDNF, act as autocrine or paracrine signals to promote neurogenesis, which ultimately results in functional recovery in neurological diseases.

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Table 1. Summary of studies investigating the effects of acupuncture or EA on neurogenesis in stroke, AD and PD with sharing symptom.

References

Species/model

Acupuncture type/ frequency/intensity/ time EA/1 or 20-Hz/muscle twitch threshold/30 min

Acupoint

Major findings

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife (GFAP+/vimentin+, GFA cells) - Upregulation of cell cyc expression

Tao et al. (2016) [15]

SD rat/cerebral ischemia (MCAO)

Ahn et al. (2016) [19]

C57BL/6 mice/cerebral ischemia (BCAS)

EA/2 Hz/2 V/20 min

Baihui (GV20), Dazhui (GV14)

- Enhancement of oligod differentiation (NG2+/B - Positive changes in the and its downstream CR

Chen et al. (2015) [66]

SD rat/cerebral ischemia (MCAO)

EA/1 or 20 Hz/below muscle contraction/30 min

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife - Upregulation of Wnt1 a

Zhao et al. (2015) [69]

SD rat/cerebral ischemia (MCAO)

EA/4 and 20 Hz/1–2 mA/15 min

Baihui (GV20), Shuigou (GV26)

- Enhancement of prolife (BrdU+/GAFP+ and Brd - Upregulation of Notch1

Fan et al. (2015) [78]

SD rat/cognition impairment (Xray irradiation)

EA/2, 15 Hz/3 mA/30 min

Baihui (GV20), Shuigou (GV26)

- EA-mediated protection synaptophysin expressio

Kim et al. (2014) [18]

C57BL/6 mice/cerebral ischemia (MCAO)

EA/2 Hz/2 V/20 min

Baihui (GV20), Dazhui (GV14)

- Enhancement of prolife (BrdU+/Dcx+, BrdU+/N - Upregulation of BDNF downstream PI3K

Huang et al. (2014) [68]

SD rat/cerebral ischemia (MCAO)

EA/1–20 Hz/6 V/30 min

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife - Promotion of cell cycle inhibitor

Luo et al. (2014) [63]

SD rat/cerebral ischemia (MCAO)

AC/none/thrust-lifted stimulation at 3 times per sec for 1 min/30 min

Shuigou (GV26)

- Enhancement of prolife - Regulation of gene tran expression

Tao et al. (2014) [43]

SD rat/cerebral ischemia (MCAO)

EA/1 or 20 Hz/muscle twitch threshold (0.01 mA)/30 min

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife - Upregulation of cell cyc - Increased BDNF and G

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APP/PS1 double Li et al. (2014) [50] transgenic mice/AD model

EA/2, 15 Hz/1 mA/30 min

Baihui (GV20)

- Decrease of Aβ deposits expression - Enhancement of prolife cells)

Yang et al. (2014) [89]

SD rat/depression (CUS)

EA/2 and 100 Hz/0.3 mA/30 min

Baihui (GV20), Yanglingquan (GB34)

- Enhancement of prolife - Enhancement of pro (BrdU+/GFAP- cells) an quiescent NP (Hoechst+

Xie et al. (2013) [67]

SD rat/cerebral ischemia (MCAO)

EA/1 and 20 Hz/None/30 min

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife - Activation of ERK path

Yang et al. (2013) [88]

SD rat/depression (CUS)

EA/2 and 100 Hz/0.3 mA/30 min

Baihui (GV20), Yanglingquan (GB34)

- Promotion of proliferati - Activation of ERK (pho

Tao et al. (2010) [65]

SD rat/cerebral ischemia (MCAO)

EA/1 and 20 Hz/muscle twitch threshold/30 min

Zusanli (ST36), Quchi (LI11)

- Enhancement of prolife and BrdU+/GFAP+ cells - No difference in the num

Cheng et al. (2009) [70]

SD rat/cerebral ischemia (MCAO)

Baihui (GV20), EA/4 Hz/2 mA/30 min Shuigou (GV26) (GV26)

Yang et al. (2008) [71]

Wistar rat/cerebral ischemia (MCAO)

EA/none/muscle showing pulsation/ 10 min

Guanyuan (CV4), - Enhancement of prolife Qihai (CV6), Brdu+/GFAP+, BrdU+/N Chengjiang (CV24)

- Enhancement of prolife - A stream-like distributio along the hippocampus, ventricle to the corpus c

- Enhancement of prolife by combination treatme

Cheng et al. (2008) [77]

SAMP8 mice/ cognition impairment

AC/none/none/none

Danzhong (CV17), Zhongwan (CV12), Qihai (CV6), Zusanli (ST36), Xuehai (SP10)

Liu et al. (2007) [87]

SD rat/depression (CUS)

EA/4 and 60 Hz/≤ 1 mA/30 min

Baihui (GV20), Anmian (EX17)

- Blocks the stress-induce (BrdU+ cells)

Kim et al. (2001) [64]

Mongolian gerbil/cerebral ischemia (CCAO)

AC/none/none/20 min

Zusanli (ST36)

- Enhancement of prolife

Abbreviations: AC, manual acupuncture; AD, Alzheimer's disease; APP, amyloid precursor protein; BCAS, bilateral common carotid artery stenosis; BDNF, brain-derived neurotrophic factor; BrdU, 5-bromo-2’-deoxyuridine; CCAO, common carotid arteries occlusion; CNPase, 2,3-cyclic nucleotide-3-phosphodiesterase; CUS, chronic unpredictable stress; CREB, cAMP 40

response element-binding protein; EA, electroacupuncture; ERK, Extracellular signalregulated kinase; GDNF, glial-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; MCAO, middle cerebral artery occlusion; NeuN, neuronal nuclei; NG2, glial antigen 2; NGF, nerve growth factor; NP, neural progenitor; Nsc, neuron specific enolase; NT4/5, neurotrophin-4/5; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3kinase; PS1, presenilin 1; SAMP8, Senescence-accelerated mouse prone 8; SD, SpragueDawley; TrkB, tyrosine receptor kinase B; VEGF, vascular endothelial growth factor.

Fig. 1.

Fig. 2.

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