A neural circuit associated with anxiety-like behaviors induced by chronic inflammatory pain and the anxiolytic effects of electroacupuncture.

AIMS: Negative emotions induced by chronic pain are a serious clinical problem. Electroacupuncture (EA) is a clinically proven safe and effective method to manage pain-related negative emotions. However, the circuit mechanisms underlying the effect of EA treatment on negative emotions remain unclear. METHODS: Plantar injection of complete Freund's adjuvant (CFA) was performed to establish a rat model of chronic inflammatory pain-induced anxiety-like behaviors. Adeno-associated virus (AAV) tracing was used to identify excitatory synaptic transmission from the rostral anterior cingulate cortex (rACC) to the dorsal raphe nucleus (DRN). Employing chemogenetic approaches, we examined the role of the rACC-DRN circuit in chronic pain-induced anxiety-like behaviors and investigated whether EA could reverse chronic pain-induced dysfunctions of the rACC-DRN circuit and anxiety-like behaviors. RESULTS: We found that chemogenetic activation of the rACC-DRN circuit alleviated CFA-induced anxiety-like behaviors, while chemogenetic inhibition of the rACC-DRN circuit resulted in short-term CFA-induced anxiety-like behaviors. Further research revealed that the development of CFA-induced anxiety-like behaviors was attributed to the dysfunction of rACC CaMKII neurons projecting to DRN serotonergic neurons (rACCCaMKII-DRN5-HT neurons) but not rACC CaMKII neurons projecting to DRN GABAergic neurons (rACCCaMKII-DRNGABA neurons). This is supported by the findings that chemogenetic activation of the rACCCaMKII-DRN5-HT circuit alleviates anxiety-like behaviors in rats with chronic pain, whereas neither chemogenetic inhibition nor chemogenetic activation of the rACCCaMKII-DRNGABA circuit altered CFA chronic pain-evoked anxiety-like behaviors in rats. More importantly, we found that EA could reverse chronic pain-induced changes in the activity of rACC CaMKII neurons and DRN 5-HTergic neurons and that chemogenetic inhibition of the rACCCaMKII-DRN5-HT circuit blocked the therapeutic effects of EA on chronic pain-induced anxiety-like behaviors. CONCLUSIONS: Our data suggest that the reversal of rACCCaMKII-DRN5-HT circuit dysfunction may be a mechanism underlying the therapeutic effect of EA on chronic pain-induced anxiety-like behaviors.

Anti-inflammatory and analgesic effect of Forsythiaside B on complete Freund's adjuvant-induced inflammatory pain in mice.

Chronic pain is frequently reported in clinical practice. Therefore, it is important to identify effective therapy to relieve pain. In this work, we selected Forsythoside B (FB), a phenylethanoid glycoside isolated from Forsythia suspensa (Thunb.) Vahl, to evaluate its effect in modulating inflammatory pain induced by complete Freund's adjuvant (CFA) and the involved mechanisms. We discovered that FB could attenuate inflammatory pain triggered by CFA injection and exert anti-anxiety effects. In detail, proinflammatory cytokines, consisting of IL-6 and TNF-α, were decreased after FB administration in the CFA-injected mice. Furthermore, the FB application ameliorated the activation of ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), the microglia and astrocytes markers respectively. Therefore, our findings indicate that FB could be a promising treatment for chronic inflammatory pain.

From 'pain management' to 'integrated health clinic': a quality improvement project to transition patients on chronic opioid therapy to more evidence-based therapies for chronic pain.

BACKGROUND: The State of New York, along with the whole nation, is struggling to combat the opioid epidemic. Major authoritative bodies on chronic pain and addiction have advocated against the use of opioids long term for chronic pain. In the spring of 2021, our pain management clinic made the decision to discontinue chronic opioid prescriptions, offering instead a three-part intervention to provide patients with support for chronic pain during the process of discontinuing chronic opioid therapy (COT). Our goal was to provide safer and more evidence-based care for our chronic pain population. OBJECTIVES: To safely wean patients in our pain management clinic off of COT and offer alternative pain interventions in order to help them reach their health goals. INTERVENTION: Our three-part intervention included a unified plan for weaning patients off COT while simultaneously offering (1) expansion of integrated pain modalities, (2) Suboxone therapy and (3) a community health worker (CHW) support programme. RESULTS: Over the course of 8 months, our clinic successfully transitioned 380 patients off of COT while simultaneously expanding access to alternative pain management modalities, Suboxone therapy and CHW support services. CONCLUSION: Alternative pain management modalities, Suboxone therapy and CHW support all help to aid patients weaning off of COT while still adequately addressing their chronic pain. Our model may be adaptable to other pain management practices hoping to decrease inappropriate use of COT.

Medical Cannabis Used as an Alternative Treatment for Chronic Pain Demonstrates Reduction in Chronic Opioid Use - A Prospective Study.

BACKGROUND: Chronic opioid therapy (COT) has been used to treat many chronic pain conditions even with poor evidence for its long-term effectiveness. Medical cannabis has emerged with certain pain-relieving properties, which has led to questions as to its' potential application, especially in relation to its effect on opioid use. OBJECTIVES: This study investigates a proposed clinical context in offering medical cannabis as a treatment for chronic pain for those already using chronic opioid therapy. It then details patients' daily morphine milligram equivalent (MME) usage. STUDY DESIGN: This single-center prospective study follows a group of patients trialing medical cannabis treatment for chronic pain that is already using COT in order to determine individual efficacy. Continued medical cannabis treatment was a decision made by the patient, after trialing medical cannabis, to either continue medical cannabis along with COT at a reduced daily MME, or to revert back to their previous COT regimen. SETTING: This study was performed at the Allegheny Health Network Institute for Pain Medicine in Pittsburgh, Pennsylvania. The state of Pennsylvania legalized medical cannabis in April of 2016, and it became available to patients in February of 2018 through medical dispensaries. METHODS: One hundred and fifteen patients met the inclusion criteria, with the majority of those excluded due to not being treated with COT. Of the 115 who chose to undergo a medical cannabis trial in addition to their COT, 75 chose to remain certified for medical cannabis as they had significant pain relief and subsequently weaned down on opioids. Additionally, of the 115 choosing to undergo a medical cannabis trial, 30 chose to be decertified due to ineffectiveness or side effects, and those were placed back on their COT regimen. The other 10 were not included for other denoted reasons. Compliance was monitored through urine drug screens (UDS). RESULTS: There was a 67.1% average decrease in daily MME/patient from 49.9 to 16.4 MME at the first follow-up. There was a 73.3% decrease in MME at second follow-up from 49.9 to 13.3 MME with an ANOVA analysis denoting a significant difference of P < 0.0001. LIMITATIONS: The period of follow-up presented at this point includes their first 6 months of treatment with medical cannabis and COT concomitantly. CONCLUSIONS: Presenting medical cannabis to chronic pain patients on COT should be done in the context of a patient choice between medical cannabis WITH decrement of COT or continued current dose of COT in order to maximize effectiveness in opioid reduction as well as to limit polypharmacy concerns regarding medical cannabis. Allowing for a temporary short-term period where patients may trial medical cannabis, while concomitantly gradually weaning their COT, is also essential in determining medical cannabis' individual effectiveness for that patient's specific type of chronic pain, which should serve to maximize long-term opioid reduction results and hence decrease opioid-related overdose deaths.

TRPV1 Responses in the Cerebellum Lobules VI, VII, VIII Using Electroacupuncture Treatment for Chronic Pain and Depression Comorbidity in a Murine Model.

Depression is a prominent complex psychiatric disorder, usually complicated through expression of comorbid conditions, with chronic pain being among the most prevalent. This comorbidity is consistently associated with a poor prognosis and has been shown to negatively impact patient outcomes. With a global rise in this condition presenting itself, the importance of discovering long-term, effective, and affordable treatments is crucial. Electroacupuncture has demonstrated renowned success in its use for the treatment of pain and is a widely recognized therapy in clinical practice for the treatment of various psychosomatic disorders, most notably depression. Our study aimed to investigate the effects and mechanisms of Acid-Saline (AS) inducing states of chronic pain and depression comorbidity in the cerebellum, using the ST36 acupoint as the therapeutic intervention. Furthermore, the role of TRPV1 was relatedly explored through the use of TRPV1-/- mice (KO). The results indicated significant differences in the four behavioral tests used to characterize pain and depression states in mice. The AS and AS + SHAM group showed significant differences when compared to the Control and AS + EA groups in the von Frey and Hargreaves's tests, as well as the Open-Field and Forced Swimming tests. This evidence was further substantiated in the protein levels observed in immunoblotting, with significant differences between the AS and AS + SHAM groups when compared to the AS + EA and AS + KO groups being identified. In addition, immunofluorescence visibly served to corroborate the quantitative outcomes. Conclusively these findings suggest that AS-induced chronic pain and depression comorbidity elicits changes in the cerebellum lobules VI, VII, VIII, which are ameliorated through the use of EA at ST36 via its action on TRPV1 and related molecular pathways. The action of TRPV1 is not singular in CPDC, which would suggest other potential targets such as acid-sensing ion channel subtype 3 (ASIC3) or voltage-gated sodium channels (Navs) that could be explored in future studies.

Therapeutic effects of Qianlie Tongli decoction on chronic prostatitis/chronic pelvic pain syndrome induced by peptide T2 in mice.

OBJECTIVES: This study was undertaken to reveal therapeutic effects and the preliminary mechanism of Chinese medicine formula Qianlie Tongli decoction (QTD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: A total of 50 male C57BL/6 mice were randomly divided into five groups. All groups except the control group were injected subcutaneously T2 peptide emulsion, which induced the CP/CPPS model. After the induction of CP/CPPS, the model group was given normal saline by oral gavage while low-dose, medium-dose and high-dose groups were treated with Chinese medicine formula. Micturition habits and pain behaviour of mice were analysed for each group. Haematoxylin and eosin (H&E) staining was used to investigate prostate inflammation. The serum level of tumour necrosis factor-α (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA) kit. KEY FINDINGS: Chinese medicine formula significantly reduced the number of urine spots and improved pain response frequency in the medium-dose and high-dose group. The high-dose group showed reduced considerably inflammatory lesion and inflammatory cell infiltration than the low-dose and medium-dose groups. Serum levels of TNF-α in the high-dose group were significantly reduced compared with the model group. CONCLUSIONS: The results demonstrated the therapeutic effects of Qianlie Tongli decoction in CP/CPPS mice by analysing clinically relevant symptoms (urinary tract system, pelvic pain and prostate inflammation) and preliminarily explored the inflammatory-related treatment mechanisms by measuring TNF-α.

A somatosensory cortex input to the caudal dorsolateral striatum controls comorbid anxiety in persistent pain.

Chronic pain and anxiety symptoms are frequently encountered clinically, but the neural circuit mechanisms underlying the comorbid anxiety symptoms in pain (CASP) in context of chronic pain remain unclear. Using viral neuronal tracing in mice, we identified a previously unknown pathway whereby glutamatergic neurons from layer 5 of the hindlimb primary somatosensory cortex (S1) (Glu), a well-known brain region involved in pain processing, project to GABAergic neurons in the caudal dorsolateral striatum (GABA). In a persistent inflammatory pain model induced by complete Freund's adjuvant injection, enhanced excitation of the Glu→GABA pathway was found in mice exhibiting CASP. Reversing this pathway using chemogenetic or optogenetic approaches alleviated CASP. In addition, the optical activation of Glu terminals in the cDLS produced anxiety-like behaviors in naive mice. Overall, the current study demonstrates the putative importance of a novel Glu→GABA pathway in controlling at least some aspects of CASP.

Epigenetic control of hypersensitivity in chronic inflammatory pain by the de novo DNA methyltransferase Dnmt3a2.

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

Opioid receptors inhibit the spinal AMPA receptor Ca2+ permeability that mediates latent pain sensitization.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (IV) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP- AMPAR antagonist IEM-1460 (50 µM) inhibited AMPAR-evoked Ca2+ transients 21d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MORCA). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MORCA inverse agonist naltrexone (NTX, 1 µg, i.t.) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 µM) increased the peak amplitude of AMPAR-evoked Ca2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain.

Chronic inflammatory pain induced GABAergic synaptic plasticity in the adult mouse anterior cingulate cortex.

Background Chronic pain is a persistent unpleasant sensation that produces pathological synaptic plasticity in the central nervous system. Both human imaging study and animal studies consistently demonstrate that the anterior cingulate cortex is a critical cortical area for nociceptive and chronic pain processing. Thus far, the mechanisms of excitatory synaptic transmission and plasticity have been well characterized in the anterior cingulate cortex for various models of chronic pain. By contrast, the potential contribution of inhibitory synaptic transmission in the anterior cingulate cortex, in models of chronic pain, is not fully understood. Methods Chronic inflammation was induced by complete Freund adjuvant into the adult mice left hindpaw. We performed in vitro whole-cell patch-clamp recordings from layer II/III pyramidal neurons in two to three days after the complete Freund adjuvant injection and examined if the model could cause plastic changes, including transient and tonic type A γ-aminobutyric acid (GABAA) receptor-mediated inhibitory synaptic transmission, in the anterior cingulate cortex. We analyzed miniature/spontaneous inhibitory postsynaptic currents, GABAA receptor-mediated tonic currents, and evoked inhibitory postsynaptic currents. Finally, we studied if GABAergic transmission-related proteins in the presynapse and postsynapse of the anterior cingulate cortex were altered. Results The complete Freund adjuvant model reduced the frequency of both miniature and spontaneous inhibitory postsynaptic currents compared with control group. By contrast, the average amplitude of these currents was not changed between two groups. Additionally, the complete Freund adjuvant model did not change GABAA receptor-mediated tonic currents nor the set of evoked inhibitory postsynaptic currents when compared with control group. Importantly, protein expression of vesicular GABA transporter was reduced within the presynpase of the anterior cingulate cortex in complete Freund adjuvant model. In contrast, the complete Freund adjuvant model did not change the protein levels of GABAA receptors subunits such as α1, α5, ß2, γ2, and δ. Conclusion Our results suggest that the induction phase of inflammatory pain involves spontaneous GABAergic plasticity at presynaptic terminals of the anterior cingulate cortex.

The Effect of a Combination of Diclofenac and Methadone Applied as Gel in a Human Experimental Pain Model - A Randomized, Placebo-controlled Trial.

Pain involves responses in which both peripheral and central mechanisms contribute to the generation of pain. Pre-clinical laboratory data have supported that a topical formulation of combined diclofenac and methadone (Diclometh) may alleviate local pain, and potentially, the side effect profile should be low. We hypothesized that antiallodynic and antihyperalgesic effects of Diclometh could be demonstrated in a human experimental pain model and that Diclometh would be safe to administer. Thus, the aims were as follows: (i) to compare two doses of Diclometh versus placebo; and (ii) to assess the safety profile of Diclometh. The study was a crossover, randomized, double-blind, placebo-controlled comparison of two doses of Diclometh gel (0.1% and 0.2%) administered topically in healthy participants. Nerve growth factor (NGF) and capsaicin intradermal injections were used as human pain models. Pressure stimulation, contact heat stimulation, hyperalgesia (pinprick stimulation) and allodynia (brush stimulation) to mechanical stimulation were performed in the area where capsaicin and NGF were injected. Side effects were recorded on a four-point Likert scale. Twenty-one men completed the study (mean age 26.14 ± 5.3). Diclometh 0.2% reduced the capsaicin-induced dynamic mechanical allodynia compared to placebo (primary end-point, p = 0.03). No other primary or secondary end-points were found significantly different (all p > 0.05). All side effects were reported as mild with no differences between treatments (p = 0.15). Indication of antiallodynic effect of Diclometh 0.2% was found. Additionally, it was demonstrated that Diclometh was safe to use.

Effects of a water extract of Lepidium meyenii root in different models of persistent pain in rats.

Lepidium meyenii (Walp.), commonly called maca, is an Andean crop belonging to the Brassicaceae family. Maca hypocotils are habitually consumed as customary food as well as traditional remedies for pathological conditions such as infertility. Moreover, the characterization of maca extracts revealed the presence of compounds that are able to modulate the nervous system. Aimed to evaluate the efficacy of L. meyenii in persistent pain, the present study analyzed the effects of a commercial root extract from maca in different animal models reproducing the most common causes of chronic painful pathologies. A qualitative characterization of this commercial extract by high performance liquid chromatography-mass spectrometry and tandem mass spectrometry analyses allowed us to confirm the presence of some macamides known as bioactive constituents of this root and the absence of the main aromatic glucosinolates. The acute oral administration of maca extract is able to reduce mechanical hypersensitivity and postural unbalance induced by the intra-articular injection of monoiodoacetate and the chronic-constriction injury of the sciatic nerve. Furthermore, L. meyenii extract reverts pain threshold alterations evoked by oxaliplatin and paclitaxel. A good safety profile in mice and rats was shown. In conclusion, the present maca extract could be considered as a therapeutic opportunity to relieve articular and neuropathic pain.

Black Tea Source, Production, and Consumption: Assessment of Health Risks of Fluoride Intake in New Zealand.

In countries with fluoridation of public water, it is imperative to determine other dietary sources of fluoride intake to reduce the public health risk of chronic exposure. New Zealand has one of the highest per capita consumption rates of black tea internationally and is one of the few countries to artificially fluoridate public water; yet no information is available to consumers on the fluoride levels in tea products. In this study, we determined the contribution of black tea as a source of dietary fluoride intake by measuring the fluoride content in 18 brands of commercially available products in New Zealand. Fluoride concentrations were measured by potentiometric method with a fluoride ion-selective electrode and the contribution of black tea to Adequate Intake (AI) and Tolerable Upper Intake Level (UL) was calculated for a range of consumption scenarios. We examined factors that influence the fluoride content in manufactured tea and tea infusions, as well as temporal changes in fluoride exposure from black tea. We review the international evidence regarding chronic fluoride intake and its association with chronic pain, arthritic disease, and musculoskeletal disorders and provide insights into possible association between fluoride intake and the high prevalence of these disorders in New Zealand.

Therapeutic effects of Qian-Yu decoction and its three extracts on carrageenan-induced chronic prostatitis/chronic pelvic pain syndrome in rats.

BACKGROUND: Qian-Yu decoction (QYD) is a traditional Chinese medicinal recipe composed of Radix astragali (Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao, Fabaceae ), Herba epimedii (Epimedium brevicornum Maxim., Berberidaceae), Herba leonuri (Leonurus japonicus Houtt., Lamiaceae), Cortex phellodendri (Phellodendron chinense Schneid., Rutaceae) and Radix achyranthis bidentatae (Achyranthes bidentata Bl., Amaranthaceae). This study aimed to evaluate the therapeutic activity of QYD against carrageenan-induced chronic prostatic/chronic pelvic pain syndrome (CP/CPPS) in rats and further elucidate its effective components. METHODS: Three types of components, total polysaccharides, total flavonoids and total saponins were separately extracted from QYD. Carrageenan-induced CP/CPPS rats were intragastrically administered with lyophilized product of QYD, individual extracts and all the combined forms of extracts for three weeks. Prostatic index (PI) was determined and histopathological analysis was performed. The levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), cyclooxygenase-2 (COX-2) and prostaglandin E2 (PEG2) in rat prostate tissues were measured using ELISA. The production of inducible nitric oxide synthase (iNOS) was evaluated by an enzymatic activity assay, and the release of nitric oxide (NO) was determined by a nitrate/nitrite assay. RESULTS: Treatment with QYD significantly ameliorated the histological changes of CP/CPPS rats and reduced the PI by 44.3%, with a marked downregulation of TNF-α (42.8% reduction), IL-1ß (45.3%), COX-2 (36.6%), PGE2 (44.2%), iNOS (54.1%) and NO (46.0%). Each of three extracts attenuated the symptom of CP/CPPS, but much more weakly than QYD. The combined administration of three extracts showed efficacy comparable to that of QYD while better than that of any combination of two extracts. A principal component analysis of the six inflammatory mediators as variables indicated that the effects of TS on CP/CPPS were rather different from those of TF and TP, which were similar. CONCLUSIONS: QYD can be beneficial in prevention and treatment of CP/CPPS. Polysaccharides, flavonoids and saponins, as the major effective components of QYD, exert a cooperative effect on CP/CPPS.

Sinomenine attenuates chronic inflammatory pain in mice.

Sinomenine, an alkaloid originally isolated from the roots of Sinomeniumacutum, is used as a traditional Chinese medicine for rheumatic arthritis. However, little is known about the neuronal mechanisms underlying the analgesic effects of sinomenine in animals with chronic inflammatory pain. In this study, we investigated the persistent inflammatory pain induced by hind paw injection of complete Freund's adjuvant (CFA) in mice, which was reversed by sinomenine administration. In the anterior cingulate cortex (ACC), a region highly associated with chronic pain processing, the upregulation of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors and Ca2+/calmodulin-dependent protein kinase II, total levels of GluA1, and phosphorylation of GluA1 at Ser831 (p-GluA1-Ser831) were reversed by systemically administrating sinomenine. Furthermore, sinomenine treatment downregulated the mammalian target of rapamycin (mTOR) pathway. Increases in p-mTOR, p-p70S6k, p-S6, and p-4EBP, which were induced by chronic inflammation, were all changed. However, sinomenine did not affect the levels of GluN2A-containing NMDA receptors and p-GluA1-Ser845, as well as the total levels of mTOR, p70S6k, S6, and 4EBP. In conclusion, results indicated that sinomenine reduced the chronic inflammatory pain induced by CFA, at least partially by regulating the GluN2B receptors and mTOR signals in the ACC.

Pulsed Radiofrequency Attenuates Complete Freund's Adjuvant-Induced Epigenetic Suppression of Potassium Chloride Cotransporter 2 Expression.

Background: Pulsed radiofrequency (PRF) treatment offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund's adjuvant (CFA)-induced inflammatory pain. Epigenetic modification of potassium-chloride cotransporter 2 (KCC2) gene expression was examined to elucidate the potential contributing mechanism. Methods: Male Sprague-Dawley rats were injected with CFA into the plantar surface of the left hind paw to induce inflammation. PRF (20 minutes of 500-kHz RF pulses, delivered at a rate of 2 Hz, maximum temperature 42ºC) was delivered to the L5 and L6 anterior primary ramus just distal to the intervertebral foramen of adult CFA or saline rats. The hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before and after CFA. Acetyl-histone H3 and H4 was determined by chromatin immunoprecipitation in spinal dorsal horn. KCC2 expression was determined by Western blot. Inhibitory synaptic function was evaluated by patch clamp in lamina II neurons. Results: KCC2 gene expression was suppressed through histone hypoacetylation, resulting in decreased efficacy of GABAergic signaling in CFA rats. PRF increased histone acetylation and KCC2 expression, partially restored the GABA synaptic function, and relieved sensitized pain behavior. Conclusion: These findings suggest that PRF might be an alternative therapy for inflammatory pain. One of the underlying mechanisms is through modification of KCC2, which is an important determinant for the efficacy of inhibitory neurotransmission in the spinal cord, and its expression levels are regulated by histone acetylation epigenetically following inflammation.

Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of ß-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty acids might have an important role in this pain control system.

The inhibition of subchondral bone lesions significantly reversed the weight-bearing deficit and the overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn in the monosodium iodoacetate induced model of osteoarthritis pain.

BACKGROUND: Chronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain. METHODS: Osteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling. RESULTS: MIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected. CONCLUSIONS: The inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.

Effects of electroacupuncture on spinal α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor in rats injected with complete Freund's adjuvant.

The effects of electroacupuncture (EA) on spinal α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor subunits in male rats injected with complete Freund's adjuvant (CFA) were investigated. Bilateral EA stimulation (2 Hz, 1 mA) was administered by needle insertion for 30 min once daily at acupoints corresponding to Zusanli and Sanyinjiao, and the thermal thresholds were measured. To examine the changes in the AMPA receptor subunits, the L4-5 segments of the spinal cord were analyzed by qPCR, western blot analysis and immunohistochemistry. The CFA-induced thermal sensitivity of the rat hind paw was significantly inhibited by EA stimulation from day 3 following CFA injection. On day 5 following CFA injection, there were no significant changes in the expression of the AMPA receptor GluR1 subunit in the CFA-injected rats with or without EA stimulation, compared with the control rats. However, the expression of the GluR2 subunit was significantly decreased by CFA treatment. Western blot analysis revealed that the expression of the phosphorylated GluR1 subunit in the control rats was not significantly different compared with that in the CFA-injected rats with and without EA stimulation. However, phosphorylation of the ipsilateral GluR2 subunit was significantly increased in the CFA-injected rats, and this activation was prevented by EA stimulation. Immunohistochemical analysis revealed a greater expression of phospho-GluR2 following CFA injection, which was inhibited by EA stimulation. These results suggested that phosphorylation of the AMPA receptor, particularly the GluR2 subunit, may be important in EA analgesia of CFA-induced inflammation.

Promoter demethylation of cystathionine-ß-synthetase gene contributes to inflammatory pain in rats.

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-ß-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.