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.
Selective targeting of sensory or nociceptive neurons in peripheral nerves remains a clinically desirable goal. Delivery of promising analgesic drugs is often impeded by the perineurium, which functions as a diffusion barrier attributable to tight junctions. We used perineurial injection of hypertonic saline as a tool to open the perineurial barrier transiently in rats and elucidated the molecular action principle in mechanistic detail: Hypertonic saline acts via metalloproteinase 9 (MMP9). The noncatalytic hemopexin domain of MMP9 binds to the low-density lipoprotein receptor-related protein-1, triggers phosphorylation of extracellular signal-regulated kinase 1/2, and induces down-regulation of the barrier-forming tight junction protein claudin-1. Perisciatic injection of any component of this pathway, including MMP9 hemopexin domain or claudin-1 siRNA, enables an opioid peptide ([D-Ala2, N -Me-Phe4,Gly5-ol]-enkephalin) and a selective sodium channel (NaV1.7)-blocking toxin (ProToxin-II) to exert antinociceptive effects without motor impairment. The latter, as well as the classic TTX, blocked compound action potentials in isolated nerves only after disruption of the perineurial barrier, which, in return, allowed endoneurally released calcitonin gene-related peptide to pass through the nerve sheaths. Our data establish the function and regulation of claudin-1 in the perineurium as the major sealing component, which could be modulated to facilitate drug delivery or, potentially, reseal the barrier under pathological conditions.
The interplay of specific leukocyte subpopulations, resident cells and proalgesic mediators results in pain in inflammation. Proalgesic mediators like reactive oxygen species (ROS) and downstream products elicit pain by stimulation of transient receptor potential (TRP) channels. The contribution of leukocyte subpopulations however is less clear. Local injection of neutrophilic chemokines elicits neutrophil recruitment but no hyperalgesia in rats. In meta-analyses the monocytic chemoattractant, CCL2 (monocyte chemoattractant protein-1; MCP-1), was identified as an important factor in the pathophysiology of human and animal pain. In this study, intraplantar injection of CCL2 elicited thermal and mechanical pain in Wistar but not in Dark Agouti (DA) rats, which lack p47phox, a part of the NADPH oxidase complex. Inflammatory hyperalgesia after complete Freund's adjuvant (CFA) as well as capsaicin-induced hyperalgesia and capsaicin-induced current flow in dorsal root ganglion neurons in DA were comparable to Wistar rats. Macrophages from DA expressed lower levels of CCR2 and thereby migrated less towards CCL2 and formed limited amounts of ROS in vitro and 4-hydroxynonenal (4-HNE) in the tissue in response to CCL2 compared to Wistar rats. Local adoptive transfer of peritoneal macrophages from Wistar but not from DA rats reconstituted CCL2-triggered hyperalgesia in leukocyte-depleted DA and Wistar rats. A pharmacological stimulator of ROS production (phytol) restored CCL2-induced hyperalgesia in vivo in DA rats. In Wistar rats, CCL2-induced hyperalgesia was completely blocked by superoxide dismutase (SOD), catalase or tempol. Likewise, inhibition of NADPH oxidase by apocynin reduced CCL2-elicited hyperalgesia but not CFA-induced inflammatory hyperalgesia. In summary, we provide a link between CCL2, CCR2 expression on macrophages, NADPH oxidase, ROS and the development CCL2-triggered hyperalgesia, which is different from CFA-induced hyperalgesia. The study further supports the impact of CCL2 and ROS as potential targets in pain therapy.