Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain.

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

Spinal 5-HT2A receptor is involved in electroacupuncture inhibition of chronic pain.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, and it is a typical disease which can develop chronic pain. Our previous study has proved that endocannabinoid (2-AG)-CB1R-GABA-5-HT pathway is involved in electroacupuncture (EA) mediated inhibition of chronic pain. However, it is still unclear which among the 5-HT receptor subtype is involved in EA evoked 5-HT mediated inhibition of chronic pain in the dorsal spinal cord. 5-HT2A is a G protein-coupled receptor and it is involved in 5-HT descending pain modulation system. We found that EA treatment at frequency of 2 Hz +1 mA significantly increased the expression of 5-HT2A receptor in the dorsal spinal cord and intrathecal injection of 5-HT2A receptor antagonist or agonist reversed or mimicked the analgesic effect of EA in each case respectively. Intrathecal injection of a selective GABAA receptor antagonist Bicuculline also reversed the EA effect on pain hypersensitivity. Additionally, EA treatment reversed the reduced expression of GABAA receptor and KCC2 in the dorsal spinal cord of KOA mice. Furthermore, we demonstrated that intrathecal 5-HT2A receptor antagonist/agonist reversed or mimicked the effect of EA up-regulate of KCC2 expression, respectively. Similarly, intrathecal injection of PLC and PKC inhibitors prevented both anti-allodynic effect and up-regulation of KCC2 expression by EA treatment. Our data suggest that EA treatment up-regulated KCC2 expression through activating 5-HT2A-Gq-PLC-PKC pathway and enhanced the inhibitory function of GABAA receptor, thereby inhibiting chronic pain in a mouse model of KOA.

5-HT7 Receptor Is Involved in Electroacupuncture Inhibition of Chronic Pain in the Spinal Cord.

Knee osteoarthritis (KOA) is a common and disabling condition characterized by attacks of pain around the joints, and it is a typical disease that develops chronic pain. Previous studies have proved that 5-HT1, 5-HT2, and 5-HT3 receptors in the spinal cord are involved in electroacupuncture (EA) analgesia. The 5-HT7 receptor plays antinociceptive role in the spinal cord. However, it is unclear whether the 5-HT7 receptor is involved in EA analgesia. The 5-HT7 receptor is a stimulatory G-protein (Gs)-coupled receptor that activates adenylyl cyclase (AC) to stimulate cyclic adenosine monophosphate (cAMP) formation, which in turn activates protein kinase A (PKA). In the present study, we found that EA significantly increased the tactile threshold and the expression of the 5-HT7 receptor in the dorsal spinal cord. Intrathecal injection of 5-HT7 receptor agonist AS-19 mimicked the analgesic effect of EA, while a selective 5-HT7 receptor antagonist reversed this effect. Moreover, intrathecal injection of AC and PKA antagonists prior to EA intervention prevented its anti-allodynic effect. In addition, GABAA receptor antagonist bicuculline administered (intrathecal, i.t.) prior to EA intervention blocked the EA effect on pain hypersensitivity. Our data suggest that the spinal 5-HT7 receptor activates GABAergic neurons through the Gs-cAMP-PKA pathway and participates in EA-mediated inhibition of chronic pain in a mouse model of KOA.

Effects of intensity of electroacupuncture on chronic pain in patients with knee osteoarthritis: a randomized controlled trial.

BACKGROUND: Conditioned pain modulation (CPM) is impaired in people with chronic pain such as knee osteoarthritis (KOA). The purpose of this randomized, controlled clinical trial was to investigate whether strong electroacupuncture (EA) was more effective on chronic pain by strengthening the CPM function than weak EA or sham EA in patients with KOA. METHODS: In this multicenter, three-arm parallel, single-blind randomized controlled trial, 301 patients with KOA were randomly assigned. Patients were randomized into three groups based on EA current intensity: strong EA (> 2 mA), weak EA (< 0.5 mA), and sham EA (non-acupoint). Treatments consisted of five sessions per week, for 2 weeks. Primary outcome measures were visual analog scale (VAS), CPM function, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). RESULTS: Three hundred one patients with KOA were randomly assigned, among which 271 (90.0%) completed the study (mean age 63.93 years old). One week of EA had a clinically important improvement in VAS and WOMAC but not in CPM function. After 2 weeks treatment, EA improved VAS, CPM, and WOMAC compared with baseline. Compared with sham EA, weak EA (3.8; 95% CI 3.45, 4.15; P < .01) and strong EA (13.54; 95% CI 13.23, 13.85; P < .01) were better in improving CPM function. Compared with weak EA, strong EA was better in enhancing CPM function (9.73; 95% CI 9.44, 10.02; P < .01), as well as in reducing VAS and total WOMAC score. CONCLUSION: EA should be administered for at least 2 weeks to exert a clinically important effect on improving CPM function of KOA patients. Strong EA is better than weak or sham EA in alleviating pain intensity and inhibiting chronic pain. TRIAL REGISTRATION: This study was registered with the Chinese Clinical Trial Registry ( ChiCTR-ICR-14005411 ), registered on 31 October 2014.

Electroacupuncture decreases Netrin-1-induced myelinated afferent fiber sprouting and neuropathic pain through µ-opioid receptors.

Purpose: We determined whether electroacupuncture (EA) reduces Netrin-1-induced myelinated primary afferent nerve fiber sprouting in the spinal cord and pain hypersensitivity associated with postherpetic neuralgia (PHN) through activation of µ-opioid receptors. Methods: PHN was induced by systemic injection of resiniferatoxin (RTX) in rats. Thirty-six days after RTX injection, a µ-opioid receptor antagonist, beta-funaltrexamine (ß-FNA) or a κ-opioid receptor antagonist, nor Binaltorphimine (nor-BNI), was injected intrathecally 30 mins before EA, once every other day for 4 times. Mechanical allodynia was tested with von Frey filaments. The protein expression level of Netrin-1 and its receptors (DCC and UNC5H2) were quantified by using western blotting. The myelinated primary afferent nerve fiber sprouting was mapped with the transganglionic tracer cholera toxin B-subunit (CTB). Results: Treatment with 2 Hz EA at "Huantiao" (GB30) and "Yanglingquan" (GB34) decreased the mechanical allodynia at 22 days and the myelinated primary afferent nerve fiber preternatural sprouting into the lamina II of the spinal dorsal horn at 42 days after RTX injection. Also, treatment with 2 Hz EA reduced the protein levels of DCC and Netrin-1 and promoted the expression of UNC5H2 in the spinal dorsal horn 42 days after RTX injection. Furthermore, the µ-opioid receptor antagonist ß-FNA, but not the κ-opioid receptor antagonist nor-BNI, reversed the effect of EA on neuropathic pain caused by RTX. In addition, morphine inhibited the Netrin-1 protein level induced by RTX in SH-SY5Y cells. Conclusions: Through activation of µ-opioid receptors, treatment with EA reduces the expression level of DCC and Netrin-1 and changes a growth-permissive environment in spinal dorsal horn into an inhibitory environment by increasing UNC5H2, thus decreasing RTX-caused primary afferent nerve sprouting in the spinal dorsal horn and neuropathic pain.

Electroacupuncture potentiates peripheral CB2 receptor-inhibited chronic pain in a mouse model of knee osteoarthritis.

PURPOSE: Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, with chronic pain as its typical symptom. Although studies have shown that an activated peripheral CB2 receptor can reduce acute pain, whether the CB2 receptor is involved in electroacupuncture (EA) inhibiting chronic pain and the involved mechanism remains unclear. The aim of this study was to investigate whether EA may strengthen peripheral CB2 receptor-inhibited chronic pain in a mouse model of KOA. MATERIALS AND METHODS: KOA was induced by intra-articular injection of monosodium iodoacetate (MIA) into the left knee joint of mice. Thermal hyperalgesia was tested with the hot plate test, and mechanical allodynia was quantified using von Frey filaments. The expression of CB2 receptor and IL-1ß were quantified by using immunofluorescence labeling. RESULTS: EA treatment at 2 Hz+1 mA significantly increased the expression of CB2 receptor in fibroblasts and decreased the expression of IL-1ß in the menisci compared with that in the KOA group. However, EA had no effect on the expression of IL-1ß in CB2-/- mice. At 2 Hz+1 mA, EA significantly increased mechanical threshold, thermal latency, and weight borne after KOA modeling. However, knockout of the CB2 receptor blocked these effects of EA. After 2 Hz+1 mA treatment, EA significantly reduced the Osteoarthritis Research Society International (OARSI) score after KOA modeling. However, EA had no significant effect on the OARSI score in CB2-/- mice. CONCLUSION: EA reduced the expression of IL-1ß by activating the CB2 receptor, thus inhibiting the chronic pain in the mouse model of KOA.

[Stimulating Frequency-dependent Analgesic Effect of Electroacupuncture via Down-regulating Expression of VEGF in Dorsal Horns of Lumbar Spinal Cord in Post Herpetic Neuralgia Rats].

OBJECTIVE: To observe the effect of different frequencies of electroacupuncture (EA) stimulation on pain threshold (PT) and expression of vascular endothelial growth factor (VEGF) in dorsal horns (DHs) of the lumbar spinal cord in resiniferatoxin (RTX)-induced post-herpetic neuralgia (PHN) rats, so as to reveal its mechanism in alleviating PHN. METHODS: Male SD rats were randomized into control, model, 2 Hz-EA, 15 Hz-EA, 100 Hz-EA and sham EA groups (n=16 in each). The PHN model was induced by a single intraperitoneal injection of RTX (250 µg/kg), and rats of the control group received intraperitoneal injection of the same dose of vehicle (10% Tween 80, 10% alcohol and 0.9% NaCl). Rats of EA treatment groups received EA stimulation (2 Hz, 15 Hz or 100 Hz, 1 mA) at the left "Huantiao" (GB 30) and "Yanglingquan" (GB 34) for 30 min, once every other day for 35 days, starting from 1 week after RTX injection. For sham control, acupuncture needles were inserted ipsilaterally into GB 30 and GB 34 for 30 min without electrical stimulation or manual needle manipulation. The mechanical allodynia was quantified with Von Frey filaments. The expression of mRNA and protein of VEGF in the DHs of lumbar spinal cord 4-6 segments (sampled under light microscope) was detected by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. RESULTS: A single RTX injection gradually induced tactile allodynia (significant reduction of the mechanical PT) within 3 weeks relevant to the control group (P<0.01). EA applied to GB 30 and GB 34 at 2 Hz and 15 Hz, but not 100 Hz, significantly decreased the tactile allodynia after the treatment (2 Hz from 2 weeks on and 15 Hz from 3 weeks on) in RTX-treated rats (P<0.05). RTX administration increased the mRNA and protein expression of VEGF in the lumbar spinal cord compared with the control group (P<0. 05). Moreover, 2 Hz, but not 15 Hz and 100 Hz EA significantly reduced VEGF mRNA and protein expression(P<0.05). The expression of both VEGF mRNA and protein was negatively correlated with mechanical PT in RTX-induced PHN rats. CONCLUSION: EA at 2 Hz can significantly reduce VEGF expression in the lumbar spinal cord DHs of PHN rats, which is possibly in part related to its effect in alleviating the mechanical allodynia. Our study suggests that 2 Hz EA is the best stimulation frequency for relieving PHN.

Electroacupuncture Potentiates Cannabinoid Receptor-Mediated Descending Inhibitory Control in a Mouse Model of Knee Osteoarthritis.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, which can lead to chronic pain. Although electroacupuncture (EA) is effective in relieving chronic pain in the clinic, the involved mechanisms remain unclear. Reduced diffuse noxius inhibitory controls (DNIC) function is associated with chronic pain and may be related to the action of endocannabinoids. In the present study, we determined whether EA may potentiate cannabinoid receptor-mediated descending inhibitory control and inhibit chronic pain in a mouse model of KOA. We found that the optimized parameters of EA inhibiting chronic pain were the low frequency and high intensity (2 Hz + 1 mA). EA reversed the reduced expression of CB1 receptors and the 2-arachidonoylglycerol (2-AG) level in the midbrain in chronic pain. Microinjection of the CB1 receptor antagonist AM251 into the ventrolateral periaqueductal gray (vlPAG) can reversed the EA effect on pain hypersensitivity and DNIC function. In addition, CB1 receptors on GABAergic but not glutamatergic neurons are involved in the EA effect on DNIC function and descending inhibitory control of 5-HT in the medulla, thus inhibiting chronic pain. Our data suggest that endocannabinoid (2-AG)-CB1R-GABA-5-HT may be a novel signaling pathway involved in the effect of EA improving DNIC function and inhibiting chronic pain.

Activation and expression of µ-calpain in dorsal root contributes to RTX-induced mechanical allodynia.

Background Calpain is a calcium-dependent cysteine protease, and inhibition of calpain by pre-treatment with MDL28170 attenuated the rat mechanical allodynia in a variety of pain models. Postherpetic neuralgia (Shingles) is a neuropathic pain conditioned with the presence of profound mechanical allodynia. Systemic injection of resiniferatoxin can reproduce the clinical symptoms of postherpetic neuralgia. In this study, we determined to study whether activation of calpain contributes to cleave the myelin basic protein of dorsal root and is involved in resiniferatoxin-induced mechanical allodynia of postherpetic neuralgia animal model. Results Resiniferatoxin up-regulated the expression and activation of µ-calpain in dorsal root. The expression of µ-calpain was located in Schwann cell of dorsal root, and resiniferatoxin increased the expression of µ-calpain in Schwann cell in L4-L6 dorsal root at six weeks after injection. Resiniferatoxin also induced myelin basic protein degradation in L4-L6 dorsal root at six weeks after injection. Moreover, intraperitoneal injection of calpain inhibitor MDL28170 prevented the degradation of myelin basic protein and then reduced the sprouting of myelinated afferent fibers into spinal lamina II, thus relieving resiniferatoxin-induced mechanical allodynia. Conclusions Up-regulation and activation of µ-calpain located in Schwann cell may be the mechanism underlying resiniferatoxin-mediated proteolysis of myelin basic protein in dorsal root. Calpain inhibitor MDL28170 prevents resiniferatoxin-induced sprouting of myelinated afferent fibers and mechanical allodynia through inhibition of degradation of the myelin basic protein in dorsal root. Our results indicate that inhibition of pathological µ-calpain activation may present an interesting novel drug target in the treatment of postherpetic neuralgia.

Netrin-1 Contributes to Myelinated Afferent Fiber Sprouting and Neuropathic Pain.

Netrin-1 is a neuronal guidance molecule implicated in the development of spinal cord neurons and cortical neurons. In the adult spinal cord, UNC5H (repulsive receptor of netrin-1), but not deleted in colorectal cancer (DCC) (attractive receptor of netrin-1), constitutes a major mode of netrin-1 signal transduction, which may be involved in axon repulsion and inhibits neurite outgrowth. Abnormal sprouting of myelinated afferent fibers in the spinal dorsal horn can cause mechanical allodynia associated with postherpetic neuralgia (PHN, Shingles) and other neuropathic pains. However, whether netrin-1 participates in sprouting of myelinated afferent fibers and mechanical allodynia remains unknown. In an ultropotent TRPV1 agonist resiniferatoxin (RTX)-induced PHN-like model, RTX treatment for 6 weeks increased netrin-1 expression in dorsal horn neurons, including NK-1-positive projection neurons. In human neuroblastoma SH-SY5Y cells, we found that TRPV1 antagonist capsazepine antagonized RTX-induced upregulation of netrin-1. After RTX treatment, UNC5H2 expression was gradually decreased, whereas DCC expression was significantly increased. Silencing netrin-1 in the spinal dorsal horn significantly attenuated RTX-induced mechanical allodynia and sprouting of myelinated fibers into the spinal lamina II. Our results suggest that RTX treatment upregulates netrin-1 expression through activation of TRPV1 receptors and change UNC5H2-rich spinal dorsal horn into a growth-permissive environment by increasing DCC expression, thus enhancing the sprouting of myelinated afferent nerves. Netrin-1 may be targeted for reducing primary afferent sprouting and mechanical allodynia in PHN and other neuropathic pain conditions.

Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced ß-Endorphin Release.

Activation of cannabinoid receptor-2 (CB2) results in ß-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gßγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced ß-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced ß-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced ß-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced ß-endorphin release through Gi/o-Gßγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists.

Efficacy of Acupuncture in Children with Nocturnal Enuresis: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Background. Nocturnal enuresis (NE) is recognized as a widespread health problem in young children and adolescents. Clinical researches about acupuncture therapy for nocturnal enuresis are increasing, while systematic reviews assessing the efficacy of acupuncture therapy are still lacking. Objective. This study aims to assess the effectiveness of acupuncture therapy for nocturnal enuresis. Materials and Methods. A comprehensive literature search of 8 databases was performed up to June 2014; randomized controlled trials which compared acupuncture therapy and placebo treatment or pharmacological therapy were identified. A meta-analysis was conducted. Results. This review included 21 RCTs and a total of 1590 subjects. The overall methodological qualities were low. The results of meta-analysis showed that acupuncture therapy was more effective for clinical efficacy when compared with placebo or pharmacological treatment. Adverse events associated with acupuncture therapy were not documented. Conclusion. Based on the findings of this study, we cautiously suggest that acupuncture therapy could improve the clinical efficacy. However, the beneficial effect of acupuncture might be overstated due to low methodological qualities. Rigorous high quality RCTs are urgently needed.

Efficacy of Acupuncture in Itch: A Systematic Review and Meta-Analysis of Clinical Randomized Controlled Trials.

Background. Itch (pruritus) is a sensitive state that provokes the desire to scratch. It is not only a common symptom of skin diseases but it also occurs in some systemic diseases. Clinical studies on the efficacy of the acupuncture therapy in alleviating itch are increasing, while systematic reviews assessing the efficacy of acupuncture therapy are still lacking. Objective. This systematic review aims to assess the effectiveness of acupuncture therapy for itch. Materials and Methods. A comprehensive literature search of eight databases was performed up to June 2014, and randomized controlled trials which compared acupuncture therapy and placebo acupuncture or no treatment group were identified. Accordingly, a meta-analysis was conducted. Results. This review included three articles of randomized controlled trials (RCTs) from a total of 2530 articles. The results of Meta-analysis showed that acupuncture therapy was effective to alleviate itch compared with placebo acupuncture and no treatment group. Conclusion. Based on the findings of this systematic review, we cautiously suggest that acupuncture therapy could improve the clinical efficacy of itch. However, this conclusion needs more studies on various ethnic samples to confirm our final conclusion.

Electroacupuncture inhibits chronification of the acute pain of knee osteoarthritis: study protocol for a randomized controlled trial.

BACKGROUND: Previous studies have shown that electroacupuncture (EA) has a significant effect on acute pain, but it has not solved the clinical problem of the chronification of acute pain. Diffuse noxious inhibitory controls (DNIC) function as a reliable indicator to predict the risk of chronic pain events. DNIC function in knee osteoarthritis (KOA) patients has been demonstrated to gradually decrease during the development of chronic pain. The purpose of this study is to conduct a randomized, controlled clinical trial to determine if EA can repair impaired DNIC function and thus prevent chronification of the acute pain of KOA. METHODS/DESIGN: This is a multicenter, single blind, randomized, controlled, three-arm, large-scale clinical trial. A total of 450 KOA patients will be randomly assigned to three groups. The strong EA group will receive EA with high-intensity current (2 mA < current < 5 mA) at the ipsilateral 'Neixiyan' (EX-LE5), 'Dubi'(ST35), 'Liangqiu'(ST34) and 'Xuehai' (SP10). The weak EA group will receive EA with low-intensity current (0 mA < current < 0.5 mA) on the same acupoints. The sham EA group will receive EA with low-intensity current (0 mA < current < 0.5 mA) with fine needles inserted superficially into the sites 2 cm lateral to the above acupoints. The patients will be treated with EA once a day, 30 minutes per session, in 5 sessions per week, for 2 weeks. In order to determine the best stage of KOA for effective EA intervention, patients within the treatment groups also will be divided into four stages. The primary outcomes are Visual Analog Scale (VAS), DNIC function and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Clinical assessments will be evaluated at baseline (before treatment) and after 5 to 10 sessions of treatment. DISCUSSION: This trial will be helpful in identifying whether strong EA is more effective than weak EA in reversing chronification of acute pain through repairing the impaired DNIC function and in screening for the best stage of KOA for effective EA intervention. TRIAL REGISTRATION: Chinese Clinical Trial Registry Number: ChiCTR-ICR-14005411. The date of registration is 31 October 2014.