Transient opening of the perineurial barrier for analgesic drug delivery.

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.

Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation.

BACKGROUND: Leukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund's adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception. RESULTS: In Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48-96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro ß-endorphin (ß-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released ß-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation. CONCLUSION: Endogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly transiently enhance pain by impairing peripheral opioid analgesia.

A peptidomimetic tight junction modulator to improve regional analgesia.

The paracellular flux of solutes through tissue barriers is limited by transmembrane tight junction proteins. Within the family of tight junction proteins, claudin-1 seems to be a key protein for tightness formation and integrity. In the peripheral nervous system, the nerve fibers are surrounded with a barrier formed by the perineurium which expresses claudin-1. To enhance the access of hydrophilic pharmaceutical agents via the paracellular route, a claudin-1 specific modulator was developed. For this purpose, we designed and investigated the claudin-1 derived peptide C1C2. It transiently increased the paracellular permeability for ions and high and low molecular weight compounds through a cellular barrier model. Structural studies revealed a ß-sheet potential for the functionality of the peptide. Perineurial injection of C1C2 in rats facilitated the effect of hydrophilic antinociceptive agents and raised mechanical nociceptive thresholds. The mechanism is related to the internalization of C1C2 and to a vesicle-like distribution within the cells. The peptide mainly colocalized with intracellular claudin-1. C1C2 decreased membrane-localized claudin-1 of cells in culture and in vivo in the perineurium of rats after perineurial injection. In conclusion, a novel tool was developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient modulation of claudin-1.

Modulation of tight junction proteins in the perineurium to facilitate peripheral opioid analgesia.

BACKGROUND: Peripheral application of opioids reduces inflammatory pain but is less effective in noninflamed tissue of rats and human patients. Hypertonic solutions can facilitate the antinociceptive activity of hydrophilic opioids in noninflamed tissue in vivo. However, the underlying mechanisms are not well understood. We hypothesized that the enhanced efficacy of opioids may be because of opening of the perineurial barrier formed by tight junction-proteins like claudin-1. METHODS: Male Wistar rats were treated intraplantarly with 10% NaCl. Pain behavior (n = 6) and electrophysiological recordings (n = 9 or more) from skin-nerve preparations after local application of the opioid [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) were explored. Tight junction-proteins as well as permeability of the barrier were examined by immunohistochemistry and Western blot (n = 3 or more). RESULTS: Local administration of 10% NaCl facilitated increased mechanical nociceptive thresholds in response to DAMGO, penetration of horseradish peroxidase into the nerve, as well as a reduced response of C- but not Aδ-nociceptors to mechanical stimulation after application of DAMGO in the skin-nerve preparation. In noninflamed paw tissue, claudin-1 was expressed in the epidermis, blood vessels, and the perineurium, surrounding neurons immunoreactive for calcitonin gene-related peptide or protein gene product 9.5. Claudin-1 but not claudin-5 or occludin was significantly reduced after pretreatment with 10% NaCl. Intraplantar application of a metalloproteinase inhibitor (GM6001) completely reversed these effects. CONCLUSION: Hypertonic saline opens the perineurial barrier via metalloproteinase activation and claudin-1 regulation, thereby allowing access of hydrophilic drugs to peripheral opioid receptors. This principle may be used to specifically target hydrophilic drugs to peripheral neurons.

Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.

Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage.

Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target.

Peripheral Interaction of Resolvin D1 and E1 with Opioid Receptor Antagonists for Antinociception in Inflammatory Pain in Rats.

Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund's adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the µ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-ß-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of ß-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate ß-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.

CXCL10 controls inflammatory pain via opioid peptide-containing macrophages in electroacupuncture.

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.

Safety, efficacy, and molecular mechanism of claudin-1-specific peptides to enhance blood-nerve-barrier permeability.

The blood-nerve barrier consists of the perineurium and endoneurial vessels. The perineurial barrier is composed of a basal membrane and a layer of perineurial cells sealed by tight junction proteins preventing e.g. application of analgesics for selective regional pain control. One of the barrier-sealing proteins in the blood-nerve barrier is claudin-1. Therefore, the claudin-1-peptidomimetics (C1C2), derived from the first extracellular loop (ECL1) on claudin-1 was developed. In this study, we further evaluated the expression of tight junction proteins in the perineurium in Wistar rats and characterized the specificity, in vivo applicability, mechanism of action as well as the biocompatibility of C1C2. In the perineurium, claudin-19, tricellulin and ZO-1, but no claudin-2, 3, 8 and -11 were expressed. C1C2 specifically bound to the ECL1 of claudin-1 and fluorescent 5,6-carboxytetramethylrhodamine-C1C2 was rapidly internalized. Opening the perineurium with C1C2 reduced the mRNA and protein expression of claudin-1 and increased small and macromolecule permeability into the peripheral nerve. Application of C1C2 facilitated regional analgesia using µ-opioid receptor agonists like DAMGO or morphine without motor impairment in naïve rats as well as rats with hind paw inflammation. In contrast the control peptide C2C2 derived from ECL1 on claudin-2 did neither open the barrier nor facilitated opioid-mediated regional analgesia. C1C2 delivery was well tolerated and caused no morphological and functional nerve damage. C1C2 effects could be reversed by interference with the wnt-signal-transduction pathway, specifically the homeobox transcription factor cdx2, using a glycogen-synthase-kinase-3 inhibitor. In summary, we describe the composition of and a pathway to open the perineurial barrier employing a peptide to deliver hydrophilic substances to the peripheral nerve.

The connection of monocytes and reactive oxygen species in pain.

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 p47(phox), 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.

The molecular link between C-C-chemokine ligand 2-induced leukocyte recruitment and hyperalgesia.

UNLABELLED: The chemokine C-C-chemokine ligand 2 (CCL2) (formerly known as MCP, macrophage chemotactic protein) is one of the important genes upregulated in different types of pain both in animals and humans. CCL2 governs the recruitment of C-C chemokine receptor 2-expressing monocytes into inflamed tissue. In contrast to neutrophilic chemokines, intraplantar injection of CCL2 in Wistar rats recruited macrophages and neutrophils and simultaneously lowered nociceptive thresholds. CCL2-induced hyperalgesia was abolished by prior systemic leukocyte depletion by cyclophosphamide and was reconstituted by local adoptive transfer of donor macrophages but not of neutrophils. Antagonists against transient receptor potential vannilloid 1 inhibited thermal and against transient receptor potential ankyrin 1 blocked mechanical hyperalgesia. Peripheral but not central activation of cyclooxygenase-2 (Cox-2) were critical for CCL2-induced hyperalgesia. In vitro CCL2 did not directly stimulate Cox-2 expression or prostaglandin E2 formation but slightly enhanced the formation of reactive oxygen species in monocytes and macrophages. In vivo, increased immunoreactivity for 4-hydroxy-2-nonenal (4-HNE), a downstream product of reactive oxygen species and known inducer of Cox-2, was observed and colocalized with Cox-2 in ED1 (CD68) positive infiltrating cells. No hyperalgesia, 4-HNE, or Cox-2 immunoreactivity was seen in leukocyte-depleted rats that were reconstituted with macrophages in the absence of CCL2, supporting the important role of CCL2. PERSPECTIVE: CCL2 plays a dual role: 1) promoting monocyte/macrophage recruitment into tissue; and 2) potentially stimulating macrophages in the tissue to produce 4-HNE and subsequently Cox-2, all resulting in the induction of hyperalgesia via transient receptor potential vannilloid 1 and transient receptor potential ankyrin 1. This encourages pharmacological efforts targeting CCL2/C-C chemokine receptor 2 and macrophages for treatment of inflammatory pain.