Mechanisms of Acupuncture-Electroacupuncture on Persistent Pain

Abstract

In the last decade, preclinical investigations of electroacupuncture mechanisms on persistent tissue-injury (inflammatory), nerve-injury (neuropathic), cancer, and visceral pain have increased. These studies show that electroacupuncture activates the nervous system differently in health than in pain conditions, alleviates both sensory and affective inflammatory pain, and inhibits inflammatory and neuropathic pain more effectively at 2–10 Hz than at 100 Hz. Electroacupuncture blocks pain by activating a variety of bioactive chemicals through peripheral, spinal, and supraspinal mechanisms. These include opioids, which desensitize peripheral nociceptors and reduce pro-inflammatory cytokines peripherally and in the spinal cord, and serotonin and norepinephrine, which decrease spinal n-methyl-d-aspartate receptor subunit GluN1 phosphorylation. Additional studies suggest that electroacupuncture, when combined with low dosages of conventional analgesics, provides effective pain management that can forestall the side effects of often-debilitating pharmaceuticals.

Introduction

Pain, a major health problem with serious social and economic consequences, costs the economy $560–635 billion annually in physician visits, analgesics, and loss of productivity.¹ Conventional medical treatments are only moderately efficacious, and they often produce problematic side effects. Acupuncture/electroacupuncture, used in China and other Asian countries for the past 3,000 yr, represents a potentially valuable adjunct to existing pain relief strategies.² Approximately two million American adults used acupuncture in 2002;³ this increased to three million in 2007, with chronic pain being the most common reason for seeking acupuncture treatment. Concomitant with increasing use of the modality, research has been performed on acupuncture mechanisms, and data from these studies have accumulated.

Based on etiology, pain may be classified into tissue damage-induced inflammatory/nociception and nerve damage-induced neuropathy. The former is caused by a painful stimulus on nociceptors, the latter by a primary lesion or dysfunction in the nervous system. Based on origin, pain may also be classified as somatic or visceral. Notably, some pain, for example cancer-related pain, experienced by one-third of patients receiving treatment for cancer and about two-thirds of those with advanced cancers, is not easily classifiable. A variety of animal models have been used to study the effect and mechanisms of electroacupuncture on persistent pain (fig. 1). This review synthesizes these studies to give an overall picture of how electroacupuncture alleviates pain through peripheral and central mechanisms of the body and to show that a number of bioactive chemicals are involved in electroacupuncture inhibition of pain.

Figure 1.

Electroacupuncture inhibits inflammatory, neuropathic, cancer, and visceral pain in various animal models.

1. Inflammatory pain animal models

The effect and mechanisms of acupuncture/electroacupuncture on persistent pain have been studied at peripheral, spinal, and supraspinal levels using inflammatory pain animal models, most of which were produced by complete Freund’s adjuvant (CFA: inactivated and dried Mycobacterium and adjuvant) or carrageenan. Many bioactive chemicals are involved in electroacupuncture inhibition of pain (table 1).

Table 1.

Bioactive Chemicals Involved in Electroacupuncture Attenuation of Persistent Pain in Animal Models

Models	Peripheral	Spinal cord	Supraspinal
Inflammatory pain	Adenosine Cannabinoids CB2R, Corticosterone COX-2, CRF IAM-1 IL-1β, IL-6, PGE2 Opioids, TNF-α	5-HT1AR, 5-HT2AR Acid-sensing ion channel 3 Akt, α2-adrenoceptors Dopamine D2 receptors Glutamate-aspartate transporter Glutamate transporter-1, GluN2A GluA1, p-GluN1, Glutamate, IL-1β, IL-6 Muscarinic cholinergic receptor Norepinephrine, Opioids, Serotonin Substance P, N/OFQ p-38 MAPK, PI3K, TNF-α	CCK-8 GABA IL-1 receptor type I Opioids
Neuropathic pain	IL-1β, IL-6, TNF-α	5-HT1AR, 5-HT3R, α2-adrenoceptors Aspartate, COX-2, extracellular signal-regulated kinases GABA, GABA receptor A, GABA receptor B GDNF, GFRα-1, Glutamate Glutamine, Glycine, IL-6, IL-1β Inducible Nitric Oxide Synthases Matrix Metallopeptidase-2/-9 Muscarinic M(1) receptors, Norepinephrine Opioids, p38 MAPK, PGE2, Serotonin Somatostatin, Superoxide anion Taurine, TNF-α
Cancer pain	β-endorphins	IL-1β, dynorphines, Substance P, TRPV1	β-endorphins
Visceral pain	β-endorphins, NK1 receptors Substance P, TNF-α, VIP VIP receptors	p38 MAPK Serotonin	5-HT, β-endorphins CRF, GluN1 Substance P

5-HT1AR = 5-hydroxytryptamine 1A receptors; 5-HT2AR = 5-hydroxytryptamine 2A receptors; CB2R = cannabinoid CB2 receptors; CCK-8 = Cholecystokinin octapeptide; COX-2 = cyclooxygenase-2; CRF = corticotrophin-releasing factor; GABA = γ-aminobutyric acid; GDNF = glial cell line-derived neurotrophic factor; GFRα-1 = GDNF family receptor α-1; IAM-1 = Intracellular adhesion molecule-1; IL-6 = interleukin-6; IL-1β = interleukin-1beta; NK-1 = neurokinin 1; N/OFQ = Nociceptin/orphanin FQ; p-38 MAPK = p-38 mitogen-activated protein kinase; PGE2 = prostaglandin E2; p-GluN1 = phosphorylated GluN1; TNF-α = tumor necrosis factor-α; TRPV1 = transient receptor potential cation channel subfamily V member 1; VIP = vasoactive intestinal polypeptides.

1.1. Peripheral mechanisms

Peripheral inflammatory cells-released opioids are involved in electroacupuncture inhibition of inflammatory pain. Studies in carrageenan-induced inflammatory pain rat models show that an intraplantar injection of naloxone or selective antagonists against μ (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2), δ (naltrindole), or κ (nor-Binaltorphimine) opioid receptors 1 h before electroacupuncture treatment at Zusanli (ST36, fig. 2) dosage-dependently blocked electroacupuncture-produced inhibition of mechanical hyperalgesia assessed through paw pressure threshold.^4,5 Consistent with these results, intraplantar naloxone methiodide, a peripherally acting opioid receptor antagonist and an antibody against β-endorphin, eliminated electroacupuncture-produced inhibition of CFA-induced thermal hyperalgesia assessed with paw withdrawal latency (PWL) in response to radiant thermal stimuli.⁶ These data indicate that electroacupuncture induces release of endogenous opioids from lymphocytes, monocytes/macrophages, and granulocytes^7,8 into inflamed skin. The opioids in turn activate receptors on peripheral nerve terminals to suppress nociception.

Figure 2.

Rat and human maps of acupoints used in pain studies.

Electroacupuncture activates sympathetic nerve fibers to increase endogenous opioid in inflammatory site. Sympathetic nerve fiber activation enhances the expression of intracellular adhesion molecule-1 in the blood vessels of inflamed tissue to promote migration of β-endorphin- and met-enkephalin-containing polymorphonuclear leukocytes and mononuclear cells in rats with CFA-induced hind paw inflammation.⁹ Further, sympathetic neuron-derived norepinephrine stimulates adrenergic receptors on inflammatory cells to release β-endorphin, leading to inhibition of pain.¹⁰ Electroacupuncture activates sympathetic nerve fibers to inhibit pain,^11,12 although the exact mechanism is not clear. For instance, pretreatment with either 6-hydroxydopamine, a neurotoxin for sympathetic nerve endings, or the β-adrenoceptor antagonist propranolol, significantly prevents electroacupuncture inhibition of carrageenan-induced thermal hyperalgesia.¹³ This electroacupuncture action on sympathetic nerves might enhance migration of opioid-containing cells to an inflammatory site, increasing the release of endogenous opioids.

Electroacupuncture also increases endogenous cannabinoid CB2 receptors (CB2R) to upregulate opioids in inflamed skin tissue. At Huantiao (GB30) and Yanglingquan (GB34), the modality significantly elevated proopiomelanocortin messenger RNA (mRNA) and β-endorphin levels in inflamed skin tissue as well as the percentage of β-endorphin-immunoreactive keratinocytes, macrophages, and T-lymphocytes.¹⁴ These effects were significantly attenuated by CB2R antagonist AM630 pretreatment. Interestingly, electroacupuncture also increased the levels of endogenous anandamide in inflamed tissue¹⁵ and the expression of CB2R on keratinocytes, macrophages, and T-lymphocytes in inflammation.¹⁶

In a recent study in the CFA-induced inflammatory pain rat model, electroacupuncture significantly increased PWL and mechanical threshold assessed with von Frey filaments and significantly decreased tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 expression in inflamed skin.¹⁷ Moreover, electroacupuncture’s inhibition of pain was significantly attenuated by the CB2R antagonist AM630, as was cytokine expression.¹⁷ Since proinflammatory cytokines facilitate nociception¹⁸ and morphine inhibits cytokine release from peripheral blood mononuclear cell cultures,^19,20 electroacupuncture mitigates pain by upregulating endogenous anandamide, which activates CB2R to promote opioid production, consequently blocking cytokine release to inhibit pain.

Electroacupuncture inhibition of cyclooxygenase-2 (COX-2) might increase levels of endogenous anandamide. The modality activated the hypothalamus-pituitary-adrenal axis to significantly increase plasma corticosterone levels in a CFA-inflammatory pain rat model^21,22 and significantly downregulated carrageenan-induced expression of COX-1, COX-2 mRNA, and their proteins.²³ It is known that the endocannabinoids anandamide and 2-arachidonoyl glycerol are metabolized by fatty acid amide hydrolase, monoacylglycerol lipase, and COX-2.²⁴ Thus electroacupuncture-induced corticosterone might inhibit COX-2 to interfere with endocannabinoid metabolism, resulting in their escalation at inflammatory sites and leading to an increase in opioids.

Corticotrophin-releasing factor (CRF) and prostaglandin E2 (PGE2) are also involved in electroacupuncture analgesia. For instance, an intraplantar CRF antagonist also prevented electroacupuncture inhibition of inflammatory pain.^6,25 CRF is known to block pain by stimulating the release of opioids from immune cells.^26,27 Thus electroacupuncture might induce skin fibroblasts to release CRF, which in turn stimulates opioid release to inhibit pain. Electroacupuncture decreased carrageenan-induced PGE2 in inflammatory paws;²³ since PGE2 receptor activation of peripheral nociceptors contributes to pain, this ability to inhibit PGE2 might also be a factor in electroacupuncture’s effect on pain.²⁸

Manual acupuncture or a local injection of the adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine at acupoint ST36 significantly inhibits mechanical allodynia and thermal hyperalgesia in wild-type but not in adenosine A1 receptor knockout mice with inflammatory and neuropathic pain.²⁹ Additionally, both interventions suppress high-intensity stimulation-evoked field excitatory postsynaptic potentials in the anterior cingulate cortex (ACC). Acupuncture also significantly increased extracellular adenosine near ST36. The investigators concluded that acupuncture attenuated the pain by increasing local adenosine that acts on A1 receptors in sensory afferents of ascending nerve tracks.²⁹

Collectively, these studies demonstrate that peripheral opioids play a central role in electroacupuncture inhibition of inflammatory pain by blocking proinflammatory cytokine release from polymorphonuclear leukocytes and mononuclear cells and by acting on peripheral opioid receptors to desensitize peripheral sensory nerves (fig. 3).

Figure 3.

Mechanisms of electroacupuncture inhibition of inflammatory and neuropathic pain. Symbols + and − respectively represent enhancement and inhibition. 5-HT1AR = 5-hydroxytryptamine 1A receptors; ACC = anterior cingulated cortex; ATF-2 = activating transcription factor-2;CB2R = Cannabinoid 2 receptor; CORT = corticosterone; COX-2 = cyclooxygenase-2; EAA = excitatory amino acid; GABA = γ-aminobutyric acid; ICAM-1 = intracellular adhesion molecule-1; IL-6 = interleukin-6; IL-1β = interleukin-1beta; LC = locus coeruleus; N/OFQ = nociceptin/orphanin FQ; NE = norepinephrine; NMDAR = n-methyl-d-aspartate receptor; NRM = nucleus raphe magnus; PAG = periaqueductal grey; p-Akt = phosphorylated Akt; PGE2 = prostaglandin E2; pGluN1 = phosphorylated GluN1; SP = substance P; TNF-α = tumor necrosis factor-α.

Electroacupuncture increases opioids at inflammatory sites via two pathways. 1) It activates sympathetic nerve fibers to enhance migration of opioid-containing cells to the site. 2) It triggers hypothalamus-pituitary-adrenal to decrease COX-2, which in turn interfere with endocannabinoid metabolism, leading to increased levels of opioids at the site. Furthermore, electroacupuncture might decrease COX-2, thus lowering PGE2 levels and alleviating pain. Electroacupuncture-upregulated endocannabinoid may directly inhibit pain because CB2 receptor activation inhibits sensory nerve activities in rat pain model.³⁰ Although peripheral CRF and adenosine are involved in electroacupuncture action, how the modality modulates their synthesis warrants further investigation.

Overall, although the current studies show that opioid, cytokines, cannabinoids, CB2R, CRF, PGE2, and adenosine are involved in acupuncture/electroacupuncture analgesia, other peripheral bioactive chemicals and receptors such as serotonin, nerve growth factor, bradykinin, and transient receptor potential channels ³¹ are implicated in inflammatory pain. Their involvement in electroacupuncture analgesia is yet to be investigated.

1.2. Spinal mechanisms

Several studies demonstrate that electroacupuncture inhibits peripheral inflammation-induced Fos expression in the spinal cord.^32–34 These indicate that electroacupuncture dampens the transmission of noxious inputs at the spinal level with the involvement of spinal opioids, serotonin (i.e., 5-hydroxytryptamine [5-HT]), norepinephrine, glutamate, glial cell/cytokines, and signal molecules.

1.2.1. Opioids

Electroacupuncture acts on spinal opioid receptors differently in acute pain and persistent inflammatory conditions. In studies with uninjured rats, low frequency (2 Hz) electroacupuncture at acupoint ST36 activated endorphin/endomorphin and enkephalin, while high frequency (100 Hz) electroacupuncture activated dynorphin to suppress nociception as assessed by the tail flick test.³⁵

In a systematic evaluation of the effect of electroacupuncture frequency, intensity, treatment duration, and pulse width in a CFA-induced inflammatory pain rat model, Dr. Lao and his colleagues found that 10 and 100 Hz electroacupuncture at GB30 both significantly increased PWL. However, the effects of treatment at 10 Hz endured longer than did those of 100 Hz³⁴ due to the greater inhibition of inflammation of the former.³⁶ That team also showed that both 10 and 100 Hz electroacupuncture-produced inhibition of thermal hyperalgesia was blocked by intrathecal μ and δ opioid receptor antagonists but not a κ opioid receptor antagonist.³⁷ Kim et al.,³⁸ in a capsaicin-induced inflammatory hind paw pain model, delivered 2 Hz of four-train pulses with 100 Hz of intra-train frequency at Houxi (SI3) and Sanyangluo (TE8) on the forelimb. This stimulation significantly raised the mechanical pain threshold of the injected paw, an effect blocked by intrathecal μ or δ opioid receptor antagonists but, again, not by a κ opioid receptor antagonist.

In a rat model produced by a carrageenan injection into the knee cavity, pretreatment with 10 Hz electroacupuncture at ST36 prior to the injection significantly improved weight-bearing force (WBF), an indication of spontaneous pain, in the injected hind limb.³⁹ This analgesic effect was blocked by an intrathecal μ-opioid receptor antagonist but not by δ or κ opioid receptor antagonists.³⁹

In a study with a rat model of ankle sprain pain, 2 Hz of four-train pulses with 100 Hz of intra-train frequency were delivered on the forelimb at Yanglao (SI6) and Hegu (LI4).⁴⁰ Electroacupuncture increased stepping force of the affected limb, and this analgesic effect was not blocked by systemic injection of the opioid antagonists naloxone or naltrexone. Using the same model, 10 Hz electroacupuncture at acupoint SI6 on the forelimb contralateral to the sprained ankle significantly improved stepping force of the sprained paw and suppressed spinal dorsal horn neural activity.⁴¹ This improvement in stepping force of the affected paw and electroacupuncture inhibition of spinal dorsal horn neural hyperactivity were not blocked by a 10 mg/kg intraperitoneal injection of the opioid receptor antagonist naltrexone, which implies that endogenous opioids are not involved in electroacupuncture-produced analgesia in walking-evoked pain in an ankle sprain pain model. However, this phenomenon needs to be confirmed with varying dosages of opioid receptor antagonist administered to the spinal cord, since different dosages of naltrexone produce distinct effects⁴² and systemic naltrexone-produced supraspinal and spinal effects might offset each other.

Collectively, these studies show that low and high frequency electroacupuncture respectively inhibit thermal pain through μ, δ, and κ opioid receptors during acute pain, but during persistent pain conditions both high and low frequencies inhibit thermal, mechanical, and spontaneous pain through μ and δ opioid receptors (table 2). The differential involvement of opioid receptors might be due to changes in sensitivity of the receptors during persistent pain. For example, dosage–response curves for intrathecally administered μ and/or δ opioid agonists, determined by PWL to noxious thermal stimuli, were shifted to the left for carrageenan-inflamed hind paws in comparison to contralateral uninflamed paws. A selective intrathecal κ receptor agonist showed no activity in this analgesic assay on either inflamed or noninflamed paws.⁴³ μ and δ but not κ receptor agonists dosage-dependently reduced mechanical hyperalgesia following repeated intramuscular injections of acid.⁴⁴ Since μ and δ receptors are distributed on both presynaptic primary afferent fibers and postsynaptic dorsal horn neurons,⁴⁵ electroacupuncture-induced opioids might inhibit the activity of noxious neurons via pre- and post-synaptic mechanisms.

Table 2.

Differential Involvement of Opioid Receptors in Electroacupuncture Attenuation of Pain during Health and in Pain Conditions

Model	EA	test	opioid receptors	Refs
Uninjured	2 Hz	Tail flick	μ	35
Uninjured	15 Hz	Tail flick	μ, δ, and κ	35
Uninjured	100 Hz	Tail flick	κ	35
Ankle sprain pain	2Hz/100 Hz	Stepping force	None	40
Ankle sprain pain	10 Hz	Stepping force	None	41
Intra-plantar CFA	10 Hz and 100 Hz	Paw withdrawal latency	μ and δ	37
Intra-plantar capsaicin	2Hz/100 Hz	Mechanical threshold	μ and δ	38
Intra-knee carrageenan	10 Hz	Weight-bearing force	μ	39
Chemotherapy Neuropathy	10 Hz	Mechanical threshold	μ, δ, and κ	136

CFA = complete Freund’s adjuvant.

The fact that electroacupuncture induces endogenous opioids to inhibit pain has clinical significance. Electroacupuncture, added to opioid therapy, might decrease the dosages required for pain control. Indeed, in our study, electroacupuncture combined with low-dosage morphine suppressed inflammatory pain better than either one did individually.³⁷

Nociceptin/orphanin FQ (N/OFQ) and opioid-like receptors play important roles in pain modulation.⁴⁶ Fu et al.⁴⁷ report that 2 and 60 Hz electroacupuncture at GB30 and GB34 significantly increased N/OFQ and opioid-like receptors in the spinal cord in a CFA-induced inflammatory pain model. Moreover, intrathecal [Nphe(1)]nociceptin(1–13)NH(2), a selective antagonist of the N/OFQ peptide receptor, significantly blocked electroacupuncture inhibition of thermal hyperalgesia, which indicates that the N/OFQ receptor mediates electroacupuncture analgesia.⁴⁸ N/OFQ has been observed in fibers and neurons in superficial laminae of the dorsal horn,⁴⁹ and it inhibits C-fiber-evoked responses and wind up.⁵⁰ This leads to the conclusion that electroacupuncture attenuates pain by inducing release of spinal N/OFQ through both pre- and post-synaptic mechanisms.

Cholecystokinin octapeptide (CCK-8), a physiological antagonist of endogenous opioids in the central nervous system, attenuates electroacupuncture analgesia. Studies in uninjured animals show that electroacupuncture produces greater analgesia in CCK-8- and CCKa receptor-deficient rats than in control animals.^51,52 Intracerebroventricular antisense CCK expression vector pSV2-CCKAS converts rats with low response to electroacupuncture and morphine into high responders,⁵³ while subcutaneous CCK(B) receptor antagonist L365,260 produces dosage-dependent (0.125–2.0 mg/kg) potentiation of analgesia induced by 100 Hz electroacupuncture.⁵⁴

Other studies show that CCKa receptors in the hypothalamus and nucleus parafascicularis and CCKb receptors in the periaqueductal grey (PAG) are associated with electroacupuncture analgesia.^55–57 Interestingly, CCKa receptor-deficient rats with neuropathic pain showed more robust response to electroacupuncture treatment than did normal rats.⁵⁸ The data lead us to hypothesize that dampened CCK function promotes acupuncture/electroacupuncture and morphine analgesia and that patients given a CCK receptor antagonist will be more sensitive to acupuncture treatment than those not given the antagonist. This warrants clinical investigation.

1.2.2. Norepinephrine and serotonin

Li et al.⁵⁹ report that electroacupuncture activated serotonin-containing nucleus raphe magnus (NRM) neurons and norepinephrine-containing locus coeruleus neurons that project to the spinal cord. That study and studies in uninjured rats^60–63 indicate that spinal serotonin and norepinephrine are involved in electroacupuncture analgesia.

Numerous studies show serotonin and norepinephrine involvement in electroacupuncture’s inhibition of pain. Silva et al.⁶⁴ used the tail flick test in uninjured rats to show that the nonselective serotonin receptor antagonist methysergide blocked both 2 and 100 Hz electroacupuncture-produced analgesia, while α1- and α2-adrenoceptors antagonists only prevented 2 Hz-induced analgesia. In an intra-knee carageenan-produced rat model, systemic administration of the nonselective adrenergic receptor antagonist yohimbine and the 5-HT3 receptor (5-HT3R) antagonist ondansetron blocked 2-Hz electroacupuncture-produced increase of weight bearing in an injured hind limb.⁶⁵ In a collagen-induced arthritis model, systemic administration of spiroxatrine, a 5-HT1A receptor (5-HT1AR) antagonist and a 5-HT3R antagonist blocked 2 Hz electroacupuncture analgesia evaluated with the tail flick test; ketanserin, a 5-HT2 receptor (5-HT2R) antagonist, did not.⁶⁶ In a behavioral and electrophysiological study in an ankle sprain rat model, 10 Hz electroacupuncture applied contralaterally to SI6 significantly improved WBF of the sprained side and suppressed spinal dorsal horn neuron activities.⁴¹ This intervention had no effect in normal rats, and the electroacupuncture effect was blocked by the alpha-adrenoceptor antagonist phentolamine. These studies clearly demonstrated that serotonin and norepinephrine are involved in electroacupuncture-produced pain inhibition but did not differentiate between spinal and supraspinal levels of antagonist action.

Administration of intrathecal antagonist demonstrated that spinal norepinephrine and serotonin are involved in electroacupuncture inhibition of pain. Intrathecal yohimbine, an α2 adrenergic antagonist, reduced 2-Hz electroacupuncture-induced analgesia in a rat model of ankle sprain, but terazosin, an α1-adrenergic antagonist, had no effect.⁶⁷ Consistent with these results, studies show that an intrathecal α2a-adrenoceptor antagonist blocked 10-Hz electroacupuncture anti-hyperalgesia in a CFA-induced inflammatory pain model, while an α2b-adrenoceptor antagonist did not.⁶⁸ These studies indicate that spinal α2a-adrenoceptors are involved in 2–10 Hz electroacupuncture analgesia in inflammatory pain. In prior studies, α2a-adrenoceptor activation diminished glutamate release from the spinal cord,⁶⁹ and group I metabotropic glutamate receptors enhanced phosphorylation of n-methyl-d-aspartate receptor (NMDAR) subunit GluN1 (NR1),⁷⁰ which modulates NMDAR activity and promotes transmission of nociceptive inputs in inflammatory pain models.⁷¹ Moreover, intrathecal clonidine, an α2-adrenoceptor agonist, significantly suppressed GluN1 phosphorylation in a pain model.⁷² Immunohistochemistry shows that α2a-adrenoceptors are located in primary afferents in the spinal cord.^68,73 Collectively, these studies show that electroacupuncture increases spinal norepinephrine to pre-synaptically decrease glutamate release, thus inhibiting GluN1 phosphorylation and pain (fig. 3).

Studies also demonstrate that 10 Hz electroacupuncture inhibits thermal hyperalgesia through spinal 5-HT1AR but not 5-HT2BR, 5-HT2CR, or 5-HT3R in a CFA inflammatory pain model,^68,74 and improves WBF through 5-HT2AR in a knee osteoarthritis pain model.⁷⁵ Investigation shows that 5-HT1AR activation prevents NMDAR GluN1 subunit phosphorylation⁷⁶ and that serotonin depletion increases nociception-evoked trigeminal NMDAR phosphorylation.⁷⁷ Immunohistochemistry shows that 5-HT1ARs are localized in GluN1-containing neurons in the spinal cord.⁷⁴ In our studies, 10 Hz electroacupuncture at GB30 inhibited CFA-upregulated p-GluN1.⁷⁸ Thus electroacupuncture inhibits pain by promoting spinal 5-HT1AR activation to post-synaptically suppress GluN1 phosphorylation (fig. 3).

It is known that pretreatment of μ receptor antagonist blocks intrathecal serotonin or clonidine-produced inhibition of inflammation-caused hyperalgesia.⁶⁸ Naloxone inhibits intrathecal 5-HT anti-nociception,⁷⁹ and selective norepinephrine reuptake inhibitors significantly increase intensity and duration of morphine anti-nociceptive activity via both α2 adrenergic and opioid receptors.⁸⁰ Isobolographic analysis revealed a synergistic interaction between an α2-adrenoceptor agonist and morphine.⁸¹ Taken together, these data show that spinal 5-HT, norepinephrine, and opioids work in concert in electroacupuncture action. However, the mechanisms of their interaction are not clear.

Clinically, serotonin and norepinephrine reuptake inhibitors and selective serotonin reuptake inhibitors are used to manage chronic pain conditions. Since electroacupuncture induces the release of spinal 5-HT and norepinephrine, electroacupuncture treatment might enhance the inhibitory effect of serotonin and norepinephrine reuptake inhibitors/selective serotonin reuptake inhibitors on pain, thus allowing pain control medication to be decreased. This is supported by a study showing that when patients were given acupuncture/electroacupuncture as an adjunct to paroxetine, an selective serotonin reuptake inhibitor, only 5.7–8.9% required a dosage increase, significantly fewer than did those given paroxetine alone (22.9%).⁸²

1.2.3. Glutamate and its receptors

Glutamate and its receptors, categorized as NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate, and metabotropic groups, are abundant in the spinal dorsal horn and play important roles in transmission of noxious messages.^83,84 In a CFA-induced pain model, studies show that electroacupuncture at 2, 15, or 120 HZ not only significantly alleviates CFA-induced mechanical hyperalgesia but also significantly decreases CFA-upregulated GluN1, GluN2A, and GluA1 in the spinal dorsal horn.⁸⁵ In dorsal root ganglia (DRG), alternation of 4 and 16 Hz electroacupuncture at GB30 and GB34 significantly inhibited CFA-induced GluN1 upregulation.⁸⁶ In our studies, 10 Hz electroacupuncture at GB30 significantly decreased CFA-enhanced GluN1 phosphorylation.⁷⁸ Collectively, these studies demonstrate that electroacupuncture inhibits transmission of noxious messages at the spinal level by dampening glutamate receptor activities.

Electroacupuncture might work through spinal norepinephrine, 5-HT, and opioids to decrease NMDAR activation and thus to inhibit pain. As mentioned in section 1.2.2, electroacupuncture might inhibit GluN1 phosphorylation by inducing spinal release of 5-HT and norepinephrine. It has been reported that microiontophoretic application of a selective μ opioid receptor agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin, reduced the NMDA-evoked responses of 100% of nociceptive specific, 93% of wide dynamic range, and 86% of low threshold neurons in the superficial and deeper dorsal horn of the medulla.⁸⁷ These studies show that electroacupuncture-induced endogenous opioids inhibit NMDA-mediated excitation in the spinal cord, thus alleviating pain. Hence electroacupuncture inhibits NMDAR by activating at least three pathways, norepinephrine, 5-HT, and opioid, thus resulting in the alleviation of pain (fig. 3).

As in the electroacupuncture plus morphine study,³⁷ electroacupuncture with low-dosage dizocilpine maleate (MK-801; a noncompetitive NMDAR antagonist) suppressed inflammatory pain better than did either modality alone.^88,89 The data from these studies once again provide a rationale for pain management and control that combine Western medicine and acupuncture, and they corroborate previously reported data: for example, etoricoxib plus acupuncture has been shown to be more effective than etoricoxib with sham acupuncture or etoricoxib alone for the treatment of knee osteoarthritis pain.⁹⁰

1.2.4. Glial cells/cytokines

Electroacupuncture inhibits spinal glial cell activation and decreases cytokines to alleviate pain. Alternating 2 and 100 Hz at GB30 and GB34 raised PWL and markedly inhibited intra-articular CFA-induced astrocyte and microglia activation and IL-1β, IL-6, and TNF-α upregulation in the spinal cord.^91–93 It is well known that spinal pro-inflammatory cytokines facilitate pain. For example, IL-1β enhances GluN1 phosphorylation to facilitate pain^94–96 and TNF-α increases NMDA currents in spinal lamina II neurons.⁹⁷ Thus electroacupuncture might inhibit glial cell activation to decrease production of pro-inflammatory cytokines, leading to the inhibition of pain. Moreover, two Hz electroacupuncture at ST36 and Sanyinjiao (SP6) once a day for three days significantly increased PWL and inhibited CFA-induced upregulation of glial fibrillary acidic protein, an astrocyte marker.⁹⁸ It also reversed CFA-caused downregulation of glutamate-aspartate transporter and glutamate transporter-1 in astrocytes. Concomitantly, proteasome inhibitor MG132 treatment significantly increased PWL and reversed CFA-induced downregulation of glutamate-aspartate transporter and glutamate transporter-1. Since electroacupuncture decreased CFA-enhanced spinal proteasome activity, it is concluded that electroacupuncture inhibition of proteasome restores glutamate transporters, leading to pain inhibition.⁹⁸

Opioid and N/OFQ peptide receptor activation might mediate electroacupuncture inhibition of cytokine synthesis in the spinal cord (fig. 3). Substance P (SP), a neuropeptide key to the transmission of noxious inputs, activates glial cells during pain.⁹⁹ Its release is blocked by μ and δ opioid receptors,¹⁰⁰ which electroacupuncture significantly activates.³⁷ Electroacupuncture also inhibits tooth pulp stimulation-evoked increase in the release of immunoreactive SP.¹⁰¹ These data indicate that electroacupuncture-induced endogenous spinal opioids decrease the release of neurotransmitters such as SP and inhibit cytokine synthesis in glial cells.

It has been reported that intrathecal administration of N/OFQ significantly downregulates CFA-exaggerated IL-1β, IL-6, and TNF-α mRNA in the spinal cord in the inflammatory pain rat model, actions which were abolished when combined with the opiate receptor-like-1 receptor-specific antagonist [Nphe(1)]N/OFQ(1–13)NH2.¹⁰² ORL1 receptors are expressed on astrocytes in the rat spinal cord. In vitro studies with astrocyte cultures also show that N/OFQ inhibits cytokine gene expression through the ORL1 receptor.¹⁰² Since electroacupuncture induces release of spinal N/OFQ,^47,48 it is plausible that electroacupuncture-induced spinal N/OFQ suppresses spinal cytokine synthesis (fig. 3).

1.2.5. Signal molecules

The influence of acupuncture on signal molecules and signal pathways has been investigated. In a carrageenan-induced inflammatory pain model, 2 Hz electroacupuncture at ST36 and SP6 significantly decreased mechanical and thermal hypersensitivity and simultaneously inhibited carrageenan-induced phosphorylation of phosphatidylinositol-3-kinase and Akt.¹⁰³ It has been reported that SP enhances Akt phosphorylation in neurokinin 1 receptor-positive neurons in laminae I–III and that phosphorylated Akt initiates and maintains inflammatory hyperalgesia.¹⁰⁴ It is well known that activation of spinal μ and δ opioid receptors potently inhibits the SP release induced by peripheral noxious stimuli.¹⁰⁰ These studies show that electroacupuncture-induced endogenous opioids inhibit SP release and Akt phosphorylation to alleviate pain (fig. 3). Moreover, in a study by Liu et al.,¹⁰⁵ 10-Hz electroacupuncture-produced inhibition of inflammatory thermal hyperalgesia was prevented when spinal Gi/o-protein function was destroyed by intrathecal pretreatment with pertussis toxin. This indicates that electroacupuncture-produced anti-hyperalgesia is mediated by pertussis toxin-sensitive Gi/o proteins and the relevant signaling pathways.

Alternating 2 Hz and 100 Hz electroacupuncture bilaterally at ST36 and Kunlun (BL60) inhibited and decreased CFA-induced pain, p38 mitogen-activated protein kinase (MAPK), and activating transcription factor-2 (ATF-2) positive cells in the dorsal spinal cord.^106,107 Activated p38 MAPK is known to play an important role during nociception and is translocated to the nucleus to phosphorylate transcriptional factors such as ATF-2.¹⁰⁸ Neurokinin-1 receptors and proinflammatory cytokines such as IL-1β and TNF-α contribute to p38 MAPK activation in the spinal cord.^109,108 Conclusively, electroacupuncture-induced inhibition of proinflammatory cytokines as detailed in section 1.2.4 and electroacupuncture inhibition of neurokinin-1 receptors¹¹⁰ inhibit p38 MAPK/ATF-2 to suppress pain (fig. 3).

Two Hz electroacupuncture at ST36 inhibited intraplantar carrageenan- and CFA-induced upregulation of acid-sensing ion channel 3 in DRG.¹¹¹ At present, it is not known how electroacupuncture modulates this channel and the relevant signal cascades.

1.2.6. Other substances

Although studies in uninjured animals have demonstrated the involvement of muscarinic, γ-aminobutyric acid (GABA), and dopamine receptors in electroacupuncture analgesia,^60,64,112 few studies have used persistent pain animal models. In an intra-knee carrageenan rat model, systemic administration of the dopamine D2 receptor antagonist metoclopropamide blocked electroacupuncture analgesia.⁶⁵ This is consistent with dopamine D2 agonist inhibition of pain.¹¹³ Further, dopamine D2 agonists enhance the anti-nociception of 5-HT and norepinephrine reuptake inhibitors.¹¹³ Electroacupuncture-induced dopamine, 5-HT, and norepinephrine might alleviate pain synergistically. In collagen-induced arthritis, systemic administration of atropine, a muscarinic cholinergic receptor antagonist, also blocked electroacupuncture analgesia.⁶⁶ Since spinal muscarinic receptors are involved in morphine- and clonidine-produced antinociceptive effects,^114,115 electroacupuncture-induced acetylcholine, opioids, and norepinephrine also might alleviate pain synergistically.

Spinal mechanisms of electroacupuncture on inflammatory pain have been extensively investigated with behavioral, molecular, and pharmacological approaches. Electroacupuncture might induce several neurotransmitters, including opioids, 5-HT, norepinephrine, dopamine, and acetylcholine, which work interactively to inhibit pain. It is unclear how electroacupuncture induces these neurotransmitters.

1.3. Supraspinal mechanisms

1.3.1. Electroacupuncture inhibition of the sensory dimension of pain

Zhao’s 2008 review⁶⁰ summarizes a number of studies in uninjured animals. Collectively, these show that many nuclei, including the NRM, PAG, locus coeruleus, arcuate, preoptic area, nucleus submedius, habenular, accumbens, caudate, septal area, and amygdala, are involved in acupuncture analgesia and that opioid peptides and their receptors in the arcuate-PAG-NRM-spinal dorsal horn pathway are pivotal in this effect.^60,116 Recently, brain mechanisms of electroacupuncture analgesia have been investigated in an inflammatory pain model. Selective μ but not κ receptor antagonists infused into the rostral ventromedial medulla (RVM) blocked 10 and 100 Hz electroacupuncture-produced antihyperalgesia assessed with PWL.¹¹⁷ Double immunofluorescence staining demonstrated that μ receptor-containing neurons are GABAnergic and that GABAa receptor-containing neurons are serotonergic in the RVM.¹¹⁷ Hence, electroacupuncture induces release of endogenous endomorphins. These activate μ opioid receptors in GABAnergic neurons that suppress GABA release. Without GABA inhibition, RVM-spinal cord projecting serotonergic neurons can be activated to inhibit inflammatory pain.⁵⁹

In a carrageenan model of inflammatory pain, 4 and 60 Hz electroacupuncture alternated at ST36 and BL60 significantly inhibited thermal hyperalgesia and carrageenan-enhanced IL-1 receptor type I mRNA expression in the PAG compared to sham control.¹¹⁸ As μ opioids and chemokine receptors interact,¹¹⁹ electroacupuncture-induced opioids might act on the latter to inhibit IL-1 receptors. Additionally, it has been reported that melanocortin 4 receptor blockage in the PAG significantly attenuated mechanical allodynia and thermal hyperalgesia and inhibited glial activation and IL-1β synthesis.¹²⁰ It is possible that electroacupuncture inhibits melanocortin 4 receptors to attenuate IL-1β and IL-1 receptor activity in the PAG, thus alleviating pain.

Compared to the spinal level, brain involvement in electroacupuncture analgesia during persistent pain has been less investigated. As pain sensation is dependent on brain function and subject to brain modulation, electroacupuncture modulation of brain might play an important role in pain inhibition. For example, deep brain stimulation of the ACC in humans significantly inhibited pain.¹²¹ Brain mechanisms of electroacupuncture analgesia warrant further investigation.

1.3.2. Electroacupuncture inhibition of the affective dimension of pain

Pain has two dimensions, the sensory/discriminative and the affective. Studies in animal pain models show that electroacupuncture inhibits the sensory dimension of pain by producing an antinociceptive effect; electroacupuncture action on the affective component has only recently been studied. A CFA inflammatory pain rat model was combined with a conditioned place avoidance test to determine whether electroacupuncture inhibits pain-induced affective response.¹²² During preconditioning, rats spent similar amounts of time in two compartments, indicating no aversion to either. After conditioning, rats that received sham electroacupuncture spent less time in a pain-paired compartment, demonstrating place aversion to that compartment. By contrast, electroacupuncture-treated rats showed no aversion to the pain-paired compartment, demonstrating that electroacupuncture inhibited the CFA-produced affective response.¹²² Saline-injected rats showed neither preference nor aversion to electroacupuncture- or sham electroacupuncture-paired chambers; this shows that the electroacupuncture treatment did not produce reward or aversion (fig. 4). Intrarostral ACC (rACC) pretreatment with a μ but not a κ opioid receptor antagonist blocked electroacupuncture inhibition of pain-related affective response.¹²² A microinjection of morphine into the ACC inhibited affective response but did not change the sensory pain threshold,¹²³ and an intrathecal μ opioid receptor antagonist blocked electroacupuncture-produced increase of PWL but not electroacupuncture inhibition of affective response.¹²² Together, these studies show that electroacupuncture induces a release of endorphins to block affective response and that this effect is not a consequence of sensory pain inhibition. Interestingly, animal studies demonstrate that several drugs, including morphine, oxycodone, tramadol, Ibuprofen, and pregabalin, show a clear dissociation between anti-aversive and anti-nociceptive potency.¹²⁴ Clinical studies found that morphine more potently attenuates the affective component than the sensory component of pain.¹²⁵ Thus electroacupuncture also might differ in its actions on the sensory and affective dimensions of pain.

Figure 4.

Electroacupuncture inhibited the affective component of pain. Conditioned place avoidance scores, used to indicate affective response, were determined by subtracting time spent in the pain-paired compartment during preconditioning from time spent in that compartment during the post-conditioning test: the less post-conditioning time spent in the compartment, the greater the affective response. * P < 0.05 compared to sham control in CFA-injected rats. Reprinted with permission from Elsevier, Eur J Pain 16(2), 2012. EA = electroacupuncture, CFA = complete Fruend’s adjuvant.

In another study, electroacupuncture attenuated formalin-induced conditioned place avoidance.¹²⁶ Further, selectively blocking of the GluN2A or GluN2B subunit of the rACC abolished intra-plantar formalin injection-induced affective pain but not the nociceptive behaviors.¹²⁷ It is known that the μ opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin significantly decreases both NMDA and non-NMDA excitatory postsynaptic potential amplitudes in nucleus accumbens neurons; this action is reversed by a μ opioid receptor antagonist.¹²⁸ Immunohistochemical data showed that NMDAR and μ receptors are colocated in rACC neurons (fig. 5). The hypothesis is that electroacupuncture-produced μ opioid receptor activation modulates NMDAR activities to attenuate pain-associated affective response (fig. 3).

Figure 5.

Microphotographs showing distribution and co-localization of GluN1 and μ opioid receptors in rACC neurons. A, B: Sections were double-labeled with guinea pig polyclonal antibody against μ opioid receptors (A, red) and goat polyclonal antibody against GluN1 (B, green). C: Merged A and B photographs showing co-localization of μ opioid receptors and GluN1 in the rACC. Arrows point to double-labeled neurons (yellow). Scale bars=50 μm. rACC = rostral anterior cingulate cortex.

In summary, electroacupuncture inhibits both sensory and affective dimensions of pain. Electroacupuncture-induced endogenous opioids in the rACC may suppress NMDAR functions, which play an important role in electroacupuncture inhibition of the affective dimension of pain. It is unclear whether other neurotransmitters, such as norepinephrine which is located in the ACC, are involved in electroacupuncture inhibition of the affective component of pain.

2. Neuropathic pain models

Although mechanisms of acupuncture analgesia have been investigated at each level of the pain pathway in inflammatory pain models, acupuncture analgesia has been studied mainly at the spinal level in neuropathic pain models¹²⁹ and shows involvement of opioids, serotonin, norepinephrine, amino acids, and glia cell/cytokines.

2.1. Opioids

Spinal opioids are involved in electroacupuncture inhibition of neuropathic pain. Systemic administration of naloxone blocked 2 Hz electroacupuncture inhibition of L5 and L6 spinal nerve ligation (SNL)- and caudal trunk nerve injury-induced neuropathic pain.^130,131 Manual acupuncture, applied at ST36 and SP6 in an SNL-induced neuropathic pain rat model, significantly reduced SNL-induced hypersensitivity; this effect was blocked by systemic nolaxone.¹³² Furthermore, administration of cumulative doses (5, 10, 20 nmol) of the μ or δ opioid receptor antagonists β-funaltrexamine hydrochloride and naltrindole blocked 2-Hz-produced electroacupuncture anti-mechanical allodynia, corroborating μ and δ opioid receptor involvement in electroacupuncture action. Cumulative doses of the κ opioid receptor antagonist nor-binaltorphimine at 3, 6, and 12 nmol did not significantly influenced electroacupuncture inhibition of pain,¹³³ possibly due to the low dosages administered. Moreover, chemotherapy-induced peripheral neuropathy is the most common and serious adverse effect of chemotherapeutic agents.¹³⁴ A paclitaxel-evoked peripheral neuropathy model has been developed using intraperitoneal 2 mg/kg/ml of paclitaxel on four alternate days (0, 2, 4, and 6).¹³⁵ Beginning on day 13, the response frequency to von Frey filaments (bending force: 4–15 g) was significantly increased in paclitaxel-injected rats compared to that in those injected with vehicle. Ten Hz electroacupuncture at GB30 significantly decreased response frequency at 4–15 g compared to sham electroacupuncture;¹³⁶ 10 Hz electroacupuncture plus a μ, δ, or κ opioid receptor antagonist did not significantly decrease mechanical hypersensitivity compared to sham electroacupuncture plus vehicle; this shows that all three antagonists blocked electroacupuncture action in this model. Clearly, μ, δ, and κ receptors are involved in 10 Hz electroacupuncture inhibition of chemotherapy-induced neuropathic pain; this is different from the opioid involvement in electroacupuncture inhibition of peripheral tissue injury-caused inflammatory pain, in which only μ and δ receptors participate. (See table 2). It has been reported that an interaction exists between μ and κ receptors: spinal μ receptor activation might promote κ receptor activation-produced antinociception.¹³⁷ Thus it is hypothesized μ and κ receptor interaction is modulated differently in neuropathic and inflammatory pain conditions. This would account for the differential involvement of opioid receptors in electroacupuncture action.

Low frequency electroacupuncture inhibits neuropathic pain more effectively than does high frequency electroacupuncture. Ten Hz electroacupuncture at GB30 significantly decreased mechanical response frequency at 4–15 g compared to sham electroacupuncture;¹³⁶ 100 Hz electroacupuncture only decreased response frequency at 15 g in a paclitaxel-evoked peripheral neuropathy model. This indicates that 10 Hz electroacupuncture inhibits mechanical allodynia/hyperalgesia more potently than 100 Hz does.¹³⁶ Similarly, 2 Hz electroacupuncture, in a caudal trunk nerve injury-induced neuropathic pain model, induced a robust and longer lasting inhibitory effect on mechanical allodynia assessed with a von Frey filament (bending force: 2 g) than did 100 Hz.¹³³ Consistent with these results, 2 Hz decreased mechanical and thermal hypersensitivity more powerfully in an L5/L6 nerve ligation-induced neuropathic pain model than did 100 Hz.¹³⁸ A single treatment at 2 Hz electroacupuncture inhibited thermal hypersensitivity for 48 h; a single treatment at 100 Hz electroacupuncture was only effective for 8 h.¹³⁸ In an electrophysiological study,¹³⁹ 2 Hz electroacupuncture induced long-term depression of C-fiber-evoked potentials in rats with SNL, while 100 Hz electroacupuncture induced long-term potential in SNL rats. Synaptic long-term depression in the spinal dorsal horn in SNL rats might be involved in the long-lasting analgesia produced by 2 HZ electroacupuncture.

2.2. Serotonin and norepinephrine

Spinal serotonin and norepinephrine participate in electroacupuncture inhibition of neuropathic pain. For example, 2 Hz electroacupuncture inhibited cold allodynia in rats with right caudal trunk resection between the S1 and S2 spinal nerves innervating the tail.¹⁴⁰ Intrathecal injection of the 5-HT1AR antagonist NAN-190 and the 5-HT3R antagonist MDL-72222 significantly blocked this effect; the 5-HT2AR antagonist ketanserin did not. Intrathecal injection of the α2-adrenoceptor antagonist yohimbine also significantly blocked electroacupuncture action, but the α1-adrenoceptor antagonist prazosin did not. These data show that spinal α2-adrenoceptors, 5-HT1ARs, and 5-HT3Rs are involved in electroacupuncture inhibition of cold allodynia in neuropathic pain. Although α2-adrenoceptors and 5-HT1ARs are involved in electroacupuncture inhibition of both inflammatory and neuropathic pain, 5-HT3Rs play a role in electroacupuncture inhibition of neuropathic but not inflammatory pain. Consistent with that activity, 5-HT3Rs are involved in spinal cord stimulation-induced analgesia during neuropathic pain.¹⁴¹ It is possible that nerve injury changes spinal 5-HT3R activities leading to the receptors’ participation in neuropathic pain modulation.

It has been reported that 2 Hz electroacupuncture inhibits nerve injury-induced GluN1 expression in the spinal cord¹³¹ and that 5-HT1ARs are located on GluN1-containing spinal cord neurons.⁶⁸ These data lead us to hypothesize that electroacupuncture activates 5-HT1ARs to inhibit NMDAR activities, thus attenuating neuropathic pain.

Although inflammatory and neuropathic pain differ, certain central spinal and brain mechanisms such as activation of NMDA receptors are common to both.¹⁴² Interestingly, electroacupuncture attenuates both inflammatory and neuropathic pain by activating spinal α2-adrenoceptors and 5-HT1ARs and inhibiting GluN1 (fig. 3).

2.3. Amino acids

Both excitatory amino acids and inhibitory amino acids play roles in electroacupuncture attenuation of neuropathic pain. In a study with a chronic constriction injury model, 2 Hz electroacupuncture significantly elevated thermal and mechanical threshold, decreased concentration of spinal glutamate, aspartate, and glutamine, and increased the contents of glycine, GABA, and taurine in the spinal cord.¹⁴³ Since this study did not employ a sham electroacupuncture control, those data need to be confirmed. Further, microdialysis was used to collect the dialysate from the spinal cord in a spared nerve injury rat model. Electroacupuncture significantly decreased dialysate glutamate compared to sham electroacupuncture.¹⁴⁴ Moreover, intrathecal administration of the GABA(A) or GABA(B) receptor antagonists gabazine and saclofen blocked electroacupuncture inhibition of cold allodynia in the rats with resected caudal trunk nerves.¹⁴⁵ Collectively, these studies demonstrate that electroacupuncture reduces the release of excitatory amino acids and promotes the release of inhibitory amino acid neurotransmitters to stop pain. In regard to excitatory amino acids, morphine decreased the formalin-enhanced release of glutamate in the spinal dorsal horn,¹⁴⁶ presynaptic 5-HT1BRs decreased glutamate release from primary afferent terminals onto medullary dorsal horn neurons,¹⁴⁷ and α2a-adrenoceptor activation diminished glutamate release from the spinal cord in previous studies.⁶⁹ Based on those studies, it is proposed that endogenous electroacupuncture-induced opioids, 5-HT, and norepinephrine suppress excitatory amino acid release (fig. 3). Regarding the inhibitory amino acid GABA, a study showed that 5-HT1ARs are expressed on GABAergic neurons.¹⁴⁸ It seems that electroacupuncture-induced 5-HT promotes the inhibitory effect of GABA to inhibit pain. Moreover, activation of δ opioid receptors reduces GABA uptake;¹⁴⁹ thus electroacupuncture-induced endogenous opioids might increase GABA in extracellular space, enhancing GABA function and leading to pain inhibition (fig. 3). Additionally, intrathecal administration of the GABA receptor A or B agonists muscimol and baclofen potentiated the antinociceptive effects of morphine.¹⁵⁰ Thus it is plausible that endogenous opioids and GABA interact during electroacupuncture treatment to alleviate pain.

2.4. Glial cells/cytokines

Glial cell and cytokines participate in electroacupuncture inhibition of neuropathic pain. For instance, two Hz electroacupuncture at ST36 significantly increased mechanical and thermal thresholds in rats with superior caudal trunk transection; it significantly inhibited nerve-damage-induced upregulation of glial fibrillary acidic protein and OX-42, matrix metallopeptidase-9 and matrix metallopeptidase-2, TNF-α, IL-6, and IL-1β in the spinal cord.¹⁵¹ In a spinal cord injury rat model, manual acupuncture at GB34 and Shuigou (DU26) significantly relieved mechanical allodynia and thermal hyperalgesia.¹⁵² Acupuncture also inhibited microglia activation, p38 MAPK and extracellular signal-regulated kinase phosphorylation, and superoxide anion production in microglia; it decreased IL-1β, IL-6, TNF-α, inducible nitric oxide synthases, COX-2, and PGE2 levels in the lumbar_4–5 spinal cord.¹⁵² In rats with tightly ligated and transected tibial and sural nerves, 1 Hz electroacupuncture at ST36 and Yinlingquan (SP9) significantly decreased nerve injury-enhanced, immune response expression of IL-1β, IL-6, and TNF-α in peripheral nerves.¹⁵³ Electroacupuncture also inhibited spinal Cox-2 in the SNL model.¹⁵⁴ These chemicals positively promote transmission of noxious inputs at the spinal level; their inhibition might contribute to electroacupuncture analgesia. As stated in 1.2.4, opioid and N/OFQ peptide receptor activation might mediate electroacupuncture inhibition of cytokine synthesis in the spinal cord. In addition, p38 MAPK and extracellular signal-regulated kinase phosphorylation mediate the development of tolerance to morphine-induced analgesia,¹⁵⁵ making electroacupuncture inhibition of this phosphorylation clinically relevant for the purpose of mitigating or delaying the development of morphine tolerance.

2.5. Other bioactive molecules

Muscarinic receptors and glial cell line-derived neurotrophic factor (GDNF) are involved in electroacupuncture action. One study demonstrates that spinal muscarinic M(1) subtype receptors mediate electroacupuncture-induced antiallodynia in neuropathic rats.¹⁵⁶ This is consistent with a report that M1 receptor activation induced analgesia in a neuropathic pain model.¹⁵⁷ Electroacupuncture significantly enhanced expression of somatostatin, GDNF, and the GDNF family receptor GFRα-1 (the high-affinity receptor of GDNF) in DRG and the spinal dorsal horn in rats with neuropathic pain.^158,159 That pretreatment with antisense oligodeoxynucleotide specifically against GFRα-1 attenuates electroacupuncture analgesia indicates that electroacupuncture enhancement of GFRα-1 contributes to analgesia.¹⁶⁰ This is supported by the fact that overexpression of GDNF in the uninjured DRG exerts analgesic effects on neuropathic pain.¹⁶¹ Interestingly, other studies showed that 5-HT induces GDNF mRNA expression in rat C6 glioma cells¹⁶² and that GDNF receptor activation significantly suppresses transient receptor potential cation channel subfamily A, number 1 channel activities that facilitate the sensation of pain.¹⁶³ We conclude that electroacupuncture-induced enhancement of serotonin activities increases GDNF synthesis, then decreases transient receptor potential cation channel subfamily A, number 1 channel activities in the spinal cord to suppress pain.

Moreover, electroacupuncture has been shown to inhibit P2×3 to attenuate neuropathic pain.¹⁶⁴ Electroacupuncture at ST36 induces nitric oxide in the gracile nucleus to attenuate pain in Zucker diabetic fatty rats.¹⁶⁵ In contrast, another study showed that electroacupuncture at ST36 and SP9 inhibits nitric oxide synthase expression in the spinal cord of neuropathic rats.¹⁶⁶ Involvement of nitric oxide in electroacupuncture analgesia needs to be confirmed.

Overall, electroacupuncture induces numerous bioactive chemicals that have inhibited neuropathic pain in several rat models (fig. 1). Whether and how it modulates supraspinal and peripheral nerve activities, which might be responsible for electroacupuncture inhibition of neuropathic pain, have not been investigated as yet.

3. Cancer pain models

Electroacupuncture has alleviated bone cancer pain in animal models. Studies in a prostate cancer pain rat model clearly show that 10 Hz electroacupuncture for 30 min a day at the equivalent of human acupoint GB 30 between days 14 and 18 after a cancer-cell injection significantly attenuated both thermal and mechanical hyperalgesia.^167,168 Moreover, electroacupuncture treatment inhibited upregulation of preprodynorphin mRNA, dynorphin, and IL-1β and its mRNA compared to sham control. Intrathecal antiserum against dynorphin A (1–17) and an IL-1 receptor antagonist significantly suppressed cancer-induced hyperalgesia. These studies show that electroacupuncture inhibition of dynorphin and IL-1β contribute to electroacupuncture analgesia in cancer pain models.^167,168

In another model, S-180 sarcoma cells were injected around the left sciatic nerve of BALB/c mice to cause mechanical allodynia.¹⁶⁹ Nine days of 2 Hz electroacupuncture at ST36 once a day for 30 min significantly inhibited thermal hyperalgesia and spontaneous pain. Electroacupuncture also inhibited SP expression in the spinal dorsal horn and increased the concentration of β-endorphin in the blood and brains of mice.¹⁶⁹ Those data warrant further confirmation since the study did not include a sham control.

In a third model, Walker 256 carcinoma cells were subcutaneously injected into the plantar region of the hind paw to cause pain,¹⁷⁰ and 2 Hz electroacupuncture, administered bilaterally at ST36, significantly decreased the induced mechanical and thermal hypersensitivity and spontaneous pain. It also markedly suppressed cancer-driven upregulation of transient receptor potential cation channel subfamily V member 1 expression in corresponding L3–5 DRG. Since transient receptor potential cation channel subfamily V member 1 facilitates cancer pain, electroacupuncture inhibition of this protein might diminish pain.¹⁷¹ Taken together, these findings indicate that electroacupuncture treatment significantly inhibits cancer pain. Since cancer-related pain is debilitating and has not been well controlled, electroacupuncture might be a useful adjuvant therapy in patients with such pain.

4. Visceral pain models

Increasing evidence shows that acupuncture effectively alleviates visceral pain in animal models through peripheral, spinal, and supraspinal mechanisms (fig. 6).

Figure 6.

Mechanisms of electroacupuncture inhibition of visceral pain. Symbols + and − respectively represent enhancement and inhibition. CRF = corticotrophin-releasing factor; DRN = dorsal raphe nucleus; NK-1 = neurokinin 1; RVM = rostral ventromedial medulla; SP = substance P; TNF-α = tumor necrosis factor-α; VIP = vasoactive intestinal polypeptides; VIPR = VIP receptor; 5-HT = 5-hydroxytryptamine

4.1. Peripheral mechanisms

Numerous peripheral chemicals including neurotransmitters, neuropeptides, and cytokines are involved in electroacupuncture inhibition of visceral pain. Alternation of 5 and100 Hz electroacupuncture bilaterally at ST36 significantly decreased visceral pain and colon 5-HT3R levels in an irritable bowel syndrome (IBS) model induced by intrarectal administration of acetic acid.¹⁷² Alternating 2 and 50 Hz electroacupuncture at ST25 and ST37 (Shangjuxu) significantly decreased colorectal distension (CRD)-induced abdominal withdrawal reflex (AWR), the number of mucosal mast cells, SP, neurokinin 1 receptors, vasoactive intestinal polypeptides (VIP), and VIP receptor expression in the colon in an IBS rat model induced by mechanical colorectal irritation.^173,174 Electroacupuncture at ST36 significantly decreased colon damage and serum and colon TNF-α mRNA expression in rats with ulcerative colitis induced by intracolon ethanol and 2,4,6-trinitrobenzenesulfonic acid.¹⁷⁵ These studies demonstrate that electroacupuncture relieves visceral pain by modulating mast cells, SP, VIP, 5-HT3Rs, and TNF-α function. In keeping with the electroacupuncture studies, a study demonstrated that blockage of 5-HT3Rs significantly increases colonic distension perception threshold in IBS patients with diarrhea.¹⁷⁶ Studies also show that SP, serotonin, and histamine, which can be released by mast cells, sensitize visceral afferents.¹⁷⁷ Patients with recurrent abdominal pain showed significantly greater expression of VIP, neurokinin 1 receptors, and TNF-α mRNA compared to control.^178,179 Thus it seems that electroacupuncture decreases numerous chemicals at peripheral sites to desensitize visceral afferents and alleviate visceral pain.

4.2. Spinal mechanisms

Opioids are involved in electroacupuncture inhibition of visceral pain. A study demonstrated that alternating 4 and 100 Hz electroacupuncture bilaterally at ST36 and ST37 significantly decreases CRD-induced AWR and the magnitude of electromyograms.¹⁸⁰ In another study, alternating 2 and 100 Hz electroacupuncture bilaterally at ST36 significantly attenuated visceral motor response to CRD and the hyperexcitability of colon DRG neurons in rats instilled with intracolonic acetic acid;¹⁸¹ this attenuation was prevented by intraperitoneal naloxone pretreatment, displaying the involvement of opioids without differentiation between peripheral, spinal, and supraspinal levels of opioid action. Pretreatment with 20 Hz electroacupuncture at Jiaji (EX-B2) significantly inhibited intracolonic formalin-caused visceral pain behavior, including abdominal licking, backward extension, contraction of the flanks, and whole body contraction.¹⁸² It suppressed p38 phosphorylation and Fos expression in the spinal cord and colon, indicating that electroacupuncture modulates spinal neuronal activities. It also increased serum β-endorphin, which might act on peripheral and central site to suppress pain. Those changes were not found in healthy electroacupuncture-treated rats,¹⁸² demonstrating that electroacupuncture modulates the nervous system differently in healthy animals and those with pain conditions. Indeed, it has been shown that patients with Bell’s palsy have distinct brain responses to acupuncture treatment compared to healthy volunteers.¹⁸³ Further, patients at different stages of Bell’s palsy varied in brain responses to acupuncture. Taken together, electroacupuncture alleviates visceral pain by modulating spinal and DRG activities through endogenous opioids. Phosphorylated p38 in microglia is known to be involved in inflammatory, neuropathic, and visceral pain.^108,184 As discussed in 1.2.4, endogenous electroacupuncture-induced opioids reduce the release of neurotransmitters such as SP and subsequently inhibit glial cell activation, which might decrease p38 phosphorylation and inhibit visceral pain.

4.3. Supraspinal mechanisms

Electroacupuncture might also attenuate visceral pain through supraspinal mechanisms. An early study showed that vasopressinergic neurons in the hypothalamic paraventricular nucleus participate in the electroacupuncture inhibition of visceral pain induced by an intraperitoneal injection of antimonium potassium tartrate.¹⁸⁵ In a gastric distension-induced pain model, 10 or 100 Hz electroacupuncture at ST36 significantly mitigated visceral pain assessed with AWR and increased hypothalamus β-endorphin and SP.¹⁸⁶ In an IBS rat model produced by mechanical colorectal irritation, alternating 2 and 50 Hz electroacupuncture at ST37 significantly inhibited visceral pain and hypothalamic CRF synthesis.¹⁸⁷ In a neonatal maternal separation stress-induced IBS rat model, 10 Hz electroacupuncture at ST36 significantly suppressed visceral hyperalgesia and inhibited Fos expression in dorsal raphe nuclei of the brainstem, the superficial dorsal horn of the spinal cord, and colonic epithelium as well as 5-HT expression in dorsal raphe nuclei and the spinal cord.¹⁸⁸

In an electrophysiological study in anesthetized healthy rats, response to CRD by convergent somatic and visceral neurons in the thalamic ventrobasal nucleus was inhibited by electrical stimulation at either the skin receptive field or acupoint ST36.¹⁸⁹ It is interesting that skin receptive fields of thalamic neurons which respond to visceral nociception lie more or less along traditional Chinese medicine’s Stomach Channel and that simulating the receptive field produces more robust inhibition than does stimulating ST36, an important point on that channel. However, it has not been determined whether the same phenomenon can be found for such behavioral tests as AWR. Moreover, alternating 5 and 100 Hz electroacupuncture at ST36 and ST37 decreased AWR and IBS-exaggerated GluN1 and Fos expression in the RVM.¹⁹⁰ Since an intra-RVM NMDAR antagonist inhibited visceral pain,¹⁹¹ electroacupuncture might relieve visceral pain by inhibiting NMDAR activation in the RVM.

Collectively, several chemicals, including β-endorphin and 5-HT, are involved in electroacupuncture inhibition of visceral pain (fig. 6), but the underlying mechanisms are not yet understood. Also, although brain structures such as the thalamic ventrobasal nucleus, the hypothalamus, paraventricular nucleus, dorsal raphe nuclei, and the RVM are involved in electroacupuncture action, how those structures work together to modulate visceral pain during electroacupuncture treatment warrants further investigation.

5. Clinical implications

The preclinical studies summarized in this review provide solid evidence that electroacupuncture treatment, compared to sham electroacupuncture, results in significant changes in bioactive chemicals--including opioids, N/OFQ, serotonin, norepinephrine, glutamate receptors and transporters, cytokines, and signal molecules--in peripheral injury sites, the spinal cord, and supraspinal structures. Findings from animal models demonstrate that electroacupuncture and sham control work through different mechanisms, which implies that patients with persistent pain might respond differently to electroacupuncture and sham controls. Indeed, electroacupuncture led to significantly higher activation at the right insula, pulvinar, and medial nucleus of the thalamus compared to sham control in patients with IBS.¹⁹²

In animal models, electroacupuncture significantly inhibits pain in comparison to sham control. Systematic reviews have shown that in clinical studies, most of which used manual acupuncture, acupuncture performed either significantly better¹⁹³ or not significantly better than sham although both improved pain score.¹⁹⁴ Interestingly, in some clinical studies, electroacupuncture produced higher pain threshold elevation¹⁹⁵ and significantly better subjective evaluations of treatment effects¹⁹⁶ than did manual acupuncture. Further, early clinical trials with electrical stimulation of acupoints in patients with osteoarthritis showed significant pain inhibition compared to sham control.^2,197,198 This is confirmed by a more recent study employing electrical stimulation of acupoints during treatment.⁹⁰ In contrast, manual acupuncture produced no significant effects compared to sham control.^199,200 Based on that evidence, we hypothesize that electroacupuncture is superior to manual acupuncture, but further investigation is warranted to confirm this premise.

The studies using electroacupuncture plus pain medication provide significant and useful information for maximizing the effect of integrative medicine in clinical settings.⁹⁰ Such combination treatments might provide effective strategies for pain management, both by enhancing treatment effectiveness and by lowering pain medication dosages thus decreasing the risk of debilitating adverse effects. For example, in an animal study, electroacupuncture combined with a subeffective dose of morphine enhanced inflammatory pain inhibition compared to morphine.³⁷ In a clinical study,²⁰¹ electroacupuncture treatment prior to surgery significantly decreased the total amount of morphine required during the first 24 h postoperation and significantly reduced the incidence of nausea and dizziness during the same period compared to sham electroacupuncture. Similarly, electroacupuncture enhanced the effects of low-dosage celecoxib on monoarthritic pain²⁰² and low-dosage indomethacin on CFA-induced inflammatory pain,²⁰³ as well as intrathecal antisense oligodeoxynucleotide to interleukin-1 receptor type I²⁰⁴ and GluA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/GluK (Kainate) receptor antagonists³³ on carrageenan-induced pain. These studies show that electroacupuncture treatment has the potential to allow the use of lower dosages of such medications in clinical settings, thus reducing side effects and improving patient quality of life.

These animal studies also show that electroacupuncture inhibition of pain is parameter dependent. Ten Hz electroacupuncture produces longer alleviation of inflammatory pain and more powerful inhibition of neuropathic pain than does 100 Hz electroacupuncture.³⁴ Electroacupuncture at 10 Hz inhibits inflammation, while electroacupuncture at 100 Hz does not.³⁶ This is useful information for designing appropriate clinical trials: we used low frequency electroacupuncture in our knee osteoarthritis clinical trial; the resulting effects were significantly better than those produced by sham control and lasted for 26 weeks.² Thus, low frequency electroacupuncture could be useful in clinical settings to manage pain.

6. Summary

In summary, the studies show that electroacupuncture significantly alleviated inflammatory, neuropathic, cancer, and visceral pain in the corresponding animal models (fig. 1). Electroacupuncture intervention activates the nervous system differently in health than in pain, as shown by the fact that opioid receptors are differentially involved in electroacupuncture inhibition of pain in healthy animals and those with persistent pain (table 2), as well as by the brain responses to acupuncture displayed by patients with Bell’s palsy compared to those of healthy volunteers.¹⁸³ Moreover, electroacupuncture parameters affect outcomes: 10 Hz electroacupuncture produces longer lasting alleviation of inflammatory pain than does 100 Hz, and 2–10 Hz inhibit nerve injury-caused allodynia/hyperalgesia more potently than does electroacupuncture at 100 Hz.

Electroacupuncture mechanisms on inflammatory pain have been extensively studied. The modality inhibits both the sensory and the affective components of inflammatory pain, acting through peripheral, spinal, and supraspinal mechanisms with the involvement of a battery of bioactive molecules including opioids, N/OFQ, serotonin, norepinephrine, glutamate receptors and transporters, cytokines, and signal molecules (table 1). Of these, opioids play a central role in electroacupuncture inhibition of all kinds of pain. Opioids desensitize peripheral nociceptors, decrease pro-inflammatory cytokines in peripheral sites, decrease cytokines and SP in the spinal cord, and are involved in electroacupuncture inhibition of affective pain. Opioids also activate the descending inhibitory system, the main neurotransmitters of which are serotonin and norepinephrine. Electroacupuncture induces serotonin and norepinephrine that in turn decrease GluN1 phosphorylation to inhibit pain (fig. 3).

Electroacupuncture mechanisms on neuropathic pain have been studied mainly at the spinal level, less so at the supraspinal level and peripheral sites. Most bioactive chemicals involved in electroacupuncture inhibition of inflammatory pain are also implicated in electroacupuncture inhibition of neuropathic pain (table 1, fig. 3).

Only a few studies have investigated the mechanisms of electroacupuncture on cancer-related pain at the spinal level, but electroacupuncture has been shown to inhibit spinal SP, IL-1β, dynorphin, and transient receptor potential cation channel subfamily V member 1 to stop cancer-caused pain. Numerous papers report that electroacupuncture inhibits visceral pain through peripheral, spinal, and supraspinal mechanisms with involvement of SP, SP receptors, VIP, and VIP receptors in the colon, 5-HT and p38 in the spinal cord, hypothalamic β-endorphin, SP, CRF, neuropeptide Y, and 5-HT in the brainstem, and NMDAR in the RVM. How these structures and bioactive molecules work together remains elusive.

Although the studies reviewed provide solid evidence for electroacupuncture analgesia, the current research has some shortcomings. First, brain mechanisms underpinning electroacupuncture analgesia are less studied than are peripheral and spinal mechanisms. Although many nuclei have been found to be involved in electroacupuncture analgesia, it is not clear how they work together in electroacupuncture inhibition of persistent pain. For example, the involvement of ACC afferents and efferents in electroacupuncture inhibition of pain has not been clarified. Given that neuronal electrical activities play important roles in the functional connection of neuronal circuits, how electroacupuncture modulates neuronal activities during persistent pain warrants investigation; imaging and electrophysiological studies may be used to explore how electroacupuncture modulates neuronal circuit connections. Second, as summarized in table 1, although many bioactive chemicals in the spinal cord participate in electroacupuncture inhibition of inflammatory and neuropathic pain, it is unclear whether these chemicals play similar roles in the brain; the peripheral mechanisms of electroacupuncture inhibition of neuropathic pain and the mechanisms of electroacupuncture inhibition of cancer pain are not well studied. Third, female rats have been seldom used in electroacupuncture analgesia studies. Since there is strong evidence for gender differences in pain and treatment outcomes,^205,206 female rats should be included in future acupuncture studies to determine whether males and females respond differently to acupuncture/electroacupuncture treatment. Clarification of acupuncture/electroacupuncture mechanisms will open a variety of opportunities to combine acupuncture/electroacupuncture with medications to manage and control pain, which makes it all the more important to continue such research.