CXCL10 controls inflammatory pain via opioid peptide-containing macrophages in electroacupuncture.
ABSTRACT: Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao--gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10--as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.
Project description:Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).Both p.o. (0.008-1.0??g·kg(-1) ) and intraplantar (0.0006??g per paw) administration of crotalphine induced antinociception in PGE2 -induced hyperalgesia. Antinociception by p.o. crotalphine (1??g·kg(-1) ) was blocked by AM630 (50??g per paw), a CB2 receptor antagonist, and by antiserum anti-dynorphin A (1??g per paw). Immunoassay studies confirmed that crotalphine increased the activation of both ?-opioid (51.7%) and CB2 (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro?EIA and blocked by AM630.Crotalphine-induced antinociception involves peripheral CB2 cannabinoid receptors and local release of dynorphin A, which is dependent on CB2 receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.
Project description:Integrative approaches such as electroacupuncture, devoid of drug effects are gaining prominence for treating pain. Understanding the mechanisms of electroacupuncture induced analgesia would benefit chronic pain conditions such as sickle cell disease (SCD), for which patients may require opioid analgesics throughout life. Mouse models are instructive in developing a mechanistic understanding of pain, but the anesthesia/restraint required to administer electroacupuncture may alter the underlying mechanisms. To overcome these limitations, we developed a method to perform electroacupuncture in conscious, freely moving, unrestrained mice. Using this technique we demonstrate a significant analgesic effect in transgenic mouse models of SCD and cancer as well as complete Freund's adjuvant-induced pain. We demonstrate a comprehensive antinociceptive effect on mechanical, cold and deep tissue hyperalagesia in both genders. Interestingly, individual mice showed a variable response to electroacupuncture, categorized into high-, moderate-, and non-responders. Mechanistically, electroacupuncture significantly ameliorated inflammatory and nociceptive mediators both peripherally and centrally in sickle mice correlative to the antinociceptive response. Application of sub-optimal doses of morphine in electroacupuncture-treated moderate-responders produced equivalent antinociception as obtained in high-responders. Electroacupuncture in conscious freely moving mice offers an effective approach to develop a mechanism-based understanding of analgesia devoid of the influence of anesthetics or restraints.
Project description:Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal ?-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the ?-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the ?-receptor antibody staining specificity. We found that the antibody specifically labeled ?-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of ?/?/?-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of ?-receptor expression in the DRG was not possible. However, we demonstrate that the ?-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, ?-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies' specificity, preferably using knockout animals.
Project description:This study evaluates the prophylactic effect of the peripherally-selective mu-opioid receptor agonist, loperamide, administered topically in a liposomal gel formulation on pain, inflammation, and disease progression in the adjuvant-induced model of experimental rheumatoid arthritis in female Lewis rats. In a randomized, blinded and controlled animal trial, AIA rats were divided into six groups consisting of eleven rats per group based on the following treatments: loperamide liposomal gel, free loperamide gel, empty liposomal gel, diclofenac gel (Voltaren®), no treatment, and naive control. Topical formulations were applied daily for a maximum of 17 days-starting from day 0 at the same time as immunization. The time course of the effect of the treatments on antinocieption and inflammation was assessed using a paw pressure analgesiometer and plethysmometer, respectively. Arthritis progression was scored daily using an established scoring protocol. At the end of the study, hind paws were processed for histological analysis. Administration of loperamide liposomal gel daily across the duration of the study produced significant peripheral antinociception as expected; however, increased the severity of inflammation and accelerated arthritis progression. This was indicated by an increase in paw volume, behavioral and observational scoring, and histological analysis compared to the control groups. In particular, histology results showed an increase in pannus formation and synovial inflammation, as well as an upregulation of markers of inflammation and angiogenesis. These findings may have implications for the use of loperamide and other opioids in arthritis and potentially other chronic inflammatory diseases.
Project description:Treatments for chronic pain are inadequate, and new options are needed. Nonpharmaceutical approaches are especially attractive with many potential advantages including safety. Light therapy has been suggested to be beneficial in certain medical conditions such as depression, but this approach remains to be explored for modulation of pain. We investigated the effects of light-emitting diodes (LEDs), in the visible spectrum, on acute sensory thresholds in naive rats as well as in experimental neuropathic pain. Rats receiving green LED light (wavelength 525 nm, 8 h/d) showed significantly increased paw withdrawal latency to a noxious thermal stimulus; this antinociceptive effect persisted for 4 days after termination of last exposure without development of tolerance. No apparent side effects were noted and motor performance was not impaired. Despite LED exposure, opaque contact lenses prevented antinociception. Rats fitted with green contact lenses exposed to room light exhibited antinociception arguing for a role of the visual system. Antinociception was not due to stress/anxiety but likely due to increased enkephalins expression in the spinal cord. Naloxone reversed the antinociception, suggesting involvement of central opioid circuits. Rostral ventromedial medulla inactivation prevented expression of light-induced antinociception suggesting engagement of descending inhibition. Green LED exposure also reversed thermal and mechanical hyperalgesia in rats with spinal nerve ligation. Pharmacological and proteomic profiling of dorsal root ganglion neurons from green LED-exposed rats identified changes in calcium channel activity, including a decrease in the N-type (CaV2.2) channel, a primary analgesic target. Thus, green LED therapy may represent a novel, nonpharmacological approach for managing pain.
Project description:Opioid-based analgesics are a major component of the lengthy pain management of burn patients, including military service members, but are problematic due to central nervous system-mediated side effects. Peripheral analgesia via targeted ablation of nociceptive nerve endings that express the transient receptor potential vanilloid channel 1 (TRPV1) may provide an improved approach. We hypothesized that local injection of the TRPV1 agonist resiniferatoxin (RTX) would produce long-lasting analgesia in a rat model of pain associated with burn injury.Baseline sensitivities to thermal and mechanical stimuli were measured in male and female Sprague-Dawley rats. Under anesthesia, a 100 °C metal probe was placed on the right hind paw for 30 seconds, and sensitivity was reassessed 72 hours following injury. Rats received RTX (0.25 μg/100 μL; ipl) into the injured hind paw, and sensitivity was reassessed across three weeks. Tissues were collected from a separate group of rats at 24 hours and/or one week post-RTX for pathological analyses of the injured hind paw, dorsal spinal cord c-Fos, and primary afferent neuropeptide immunoreactivity.Local RTX reversed burn pain behaviors within 24 hours, which lasted through recovery at three weeks. At one week following RTX, decreased c-Fos and primary afferent neuropeptide immunoreactivities were observed in the dorsal horn, while plantar burn pathology was unaltered.These results indicate that local RTX induces long-lasting analgesia in a rat model of pain associated with burn. While opioids are undesirable in trauma patients due to side effects, RTX may provide valuable long-term, nonopioid analgesia for burn patients.
Project description:The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using mu-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hind paw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hind paw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected complete Freund's adjuvant (CFA) in the hind paw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hind paw. These results show that acute inflammation, but not chronic inflammation, induces segmental opioid release in the spinal cord that involves supraspinal signals.
Project description:Low-level laser acupuncture (LLLA) produces photobiomodulation through acupuncture point and is an alternative to low-level laser therapy. Although the analgesic effect of LLLA on chronic pain has been proven, its effect on acute postincisional pain has yet to be investigated. A plantar incision (PI) model was used to mimic human postsurgical pain. Male adult rats received GaAlAs laser irradiation at the right ST36 acupoint immediately after operation and on the following 4 days. Three laser treatment groups (two red laser groups with a 30- or 15-min treatment duration and one 30-min near-infrared laser group) were compared with sham LLLA and naive groups and an electroacupuncture (EA) group (separate study). Behavioral withdrawal thresholds of both hind paws were measured before and after incision. Expression of mitogen-activated protein kinases (p-ERK and p-p38), inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNF) in the spinal cord was analyzed. All three LLLA treatments attenuated post-PI tactile allodynia in the ipsilateral paw, but only the 30-min red laser treatment affected the contralateral paw and had similar efficacy to that of EA. All laser treatments barely reduced heat hyperalgesia in both hind paws. At 3 days after PI, the 30-min red laser group showed reversed increases of PI-induced p-ERK, p-p38, and iNOS but not TNF expression in the spinal cord. Repetitive LLLA treatments ameliorated PI-induced mechanical pain. The inhibition of multiple sensitization signals highlights the unique clinical role of LLLA. Thus, LLLA is an alternative to EA as an adjuvant for postoperative pain control.
Project description:BACKGROUND AND PURPOSE The endocannabinoid 2-arachidonoylglycerol (2-AG) is degraded primarily by monoacylglycerol lipase (MGL). We compared peripheral antinociceptive effects of JZL184, a novel irreversible MGL inhibitor, with the reversible MGL-preferring inhibitor URB602 and exogenous 2-AG in rats. EXPERIMENTAL APPROACH Nociception in the formalin test was assessed in groups receiving dorsal paw injections of vehicle, JZL184 (0.001-300 µg), URB602 (0.001-600 µg), 2-AG (ED(50)), 2-AG + JZL184 (at their ED(50)), 2-AG + URB602 (at their ED(50)), AM251 (80 µg), AM251 + JZL184 (10 µg), AM630 (25 µg) or AM630 + JZL184 (10 µg). Effects of MGL inhibitors on endocannabinoid accumulation and on activities of endocannabinoid-metabolizing enzymes were assessed. KEY RESULTS Intra-paw administration of JZL184, URB602 and 2-AG suppressed early and late phases of formalin pain. JZL184 and URB602 acted through a common mechanism. JZL184 (ED(50) Phase 1: 0.06 ± 0.028; Phase 2: 0.03 ± 0.011 µg) produced greater antinociception than URB602 (ED(50) Phase 1: 120 ± 51.3; Phase 2: 66 ± 23.9 µg) or 2-AG. Both MGL inhibitors produced additive antinociceptive effects when combined with 2-AG. Antinociceptive effects of JZL184, like those of URB602, were blocked by cannabinoid receptor 1 (CB(1)) and cannabinoid receptor 2 (CB(2)) antagonists. JZL184 suppressed MGL but not fatty-acid amide hydrolase or N-arachidonoyl-phosphatidylethanolamine phospholipase D activities ex vivo. URB602 increased hind paw 2-AG without altering anandamide levels. CONCLUSIONS AND IMPLICATIONS MGL inhibitors suppressed formalin-induced pain through peripheral CB(1) and CB(2) receptor mechanisms. MGL inhibition increased paw skin 2-AG accumulation to mediate these effects. MGL represents a target for the treatment of inflammatory pain.
Project description:Cannabinoid CB(2) receptors represent a therapeutic target that circumvents unwanted central side effects (e.g., psychoactivity and/or addiction) associated with activation of CB(1) receptors. One of the primary investigative tools used to study functions of the CB(2) receptor is the aminoalkylindole (R,S)-AM1241. However, (R,S)-AM1241 has been described as an atypical CB(2) agonist which produces antinociception mediated indirectly by opioid receptors. (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, were evaluated for antinociception in response to thermal (Hargreaves) and mechanical (von Frey) stimulation. Pharmacological specificity was established using antagonists for CB(1) (rimonabant [SR141716]) and CB(2) (SR144528). The opioid antagonist naloxone was administered locally in the paw or systemically to evaluate the contribution of opioid receptors to CB(2)-mediated antinociception produced by (R,S)-AM1241, (R)-AM1241, and (S)-AM1241. Comparisons were made with the opioid analgesic morphine. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 (0.033-10 mg/kg i.p.) produced antinociception to thermal, but not mechanical, stimulation of the hindpaw in naive rats. Antinociception produced by (R,S)-AM1241 and (S)-AM1241 exhibited an inverted U-shaped dose response curve. (R)-AM1241 produced greater antinociception than either (S)-AM1241 or (R,S)-AM1241 at the lowest (0.033 and 0.1 mg/kg i.p.) and highest (10 mg/kg i.p.) doses. Similar levels of antinociception were observed at intermediate doses. (R,S)-AM1241, (R)-AM1241, and (S)-AM1241 each produced CB(2)-mediated antinociception that was blocked by SR144528 but not by rimonabant. Local and systemic naloxone blocked morphine-induced antinociception but did not block antinociceptive effects of (R,S)-AM1241, (R)-AM1241, or (S)-AM1241. The antinociceptive effects of the CB(2)-selective cannabinoid (R,S)-AM1241 and its enantiomers, (R)-AM1241 and (S)-AM1241, are not dependent upon opioid receptors.