Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the ␤ chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3͞L6) with compressed lumbar DRG (L4͞L5) for changes in the gene expression of CCR2. In situ hybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4͞L5 and ipsilateral noncompressed L3͞L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 ؎ 3.5%, 49 ؎ 6.2%, and 41 ؎ 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4͞L5 and noncompressed L3͞L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRG in vitro produced potent excitatory effects not observed in control ganglia. MCP-1͞CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.hyperalgesia ͉ nerve injury ͉ neuropathic pain ͉ peripheral sensitization I nf lammation accompanying peripheral nerve injury frequently produces neuropathic pain symptoms, such as hyperalgesia and allodynia. This hyperalgesia may reflect ongoing or ectopic changes in the excitability of neurons in both injured and adjacent uninjured dorsal root ganglion (DRG) (1). Mechanisms that may contribute to the changes in neuronal activity include altered expression of ion channels, kinases, enzymes, neuropeptides, transcription factors, neurotrophins, and͞or the de novo presence of proinflammatory mediators such as cytokines, chemokines, and their respective receptors. However, current knowledge of the modification of molecular properties in both injured and noninjured adjacent ganglia is limited.Recent studies implicate the ␤ chemokine receptor 2 (CCR2) in the development and maintenance of pain (2-4). CCR2 is a G protein-coupled receptor that is related in structure to other CCRs (5, 6) and is largely thought to be a major regulator of induced macrophage migration (7-9). CCR2 is also constitutively expressed by different types of cells in the central nervous system, including neurons (10, 11), activated astrocytes (12, 13), microglia (3), and neural progenitor cells (14,15).Most CCRs, including CCR2, bind multiple chemokines (16, 17). CCR2 binds a family of closely related ␤-chemokines (C-C) called monocyte chemoattractant proteins (MCP), of whic...

Chronic pain hypersensitivity depends on N-type voltage-gated calcium channels (CaV2.2). However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects that result from inhibited physiological functions of these channels. Here we report suppression of both inflammatory and neuropathic hypersensitivity by inhibiting the binding of the axonal collapsin response mediator protein 2 (CRMP-2) to CaV2.2, thus reducing channel function. A 15-amino acid peptide of CRMP-2 fused to the transduction domain of HIV TAT protein (TAT-CBD3) decreases neurotransmitter release from nociceptive dorsal root ganglion neurons, reduces meningeal blood flow, reduces nocifensive behavior induced by subcutaneous formalin injection or following corneal capsaicin application, and reverses neuropathic hypersensitivity produced by the antiretroviral drug 2’,3’-dideoxycytidine. TAT-CBD3 was mildly anxiolytic but innocuous on sensorimotor and cognitive functions and despair. By preventing CRMP-2-mediated enhancement of CaV2.2 function, TAT-CBD3 alleviates inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing clinical pain.

BackgroundAnimal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.ResultsFocal nerve demyelination increased behavioral reflex responsiveness to mechanical stimuli between postoperative day (POD) 3 and POD28 in both the hindpaw ipsilateral and contralateral to the nerve injury. This behavior was accompanied by a bilateral increase in the numbers of primary sensory neurons expressing the chemokine receptors CCR2, CCR5, and CXCR4 by POD14, with no change in the pattern of CXCR3 expression. Significant increases in the numbers of neurons expressing the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), Regulated on Activation, Normal T Expressed and Secreted (RANTES/CCL5) and interferon γ-inducing protein-10 (IP-10/CXCL10) were also evident following nerve injury, although neuronal expression pattern of stromal cell derived factor-1α (SDF1/CXCL12) did not change. Functional studies demonstrated that acutely dissociated sensory neurons derived from LPC-injured animals responded with increased [Ca2+]i following exposure to MCP-1, IP-10, SDF1 and RANTES on POD 14 and 28, but these responses were largely absent by POD35. On days 14 and 28, rats received either saline or a CCR2 receptor antagonist isomer (CCR2 RA-[R]) or its inactive enantiomer (CCR2 RA-[S]) by intraperitoneal (i.p.) injection. CCR2 RA-[R] treatment of nerve-injured rats produced stereospecific bilateral reversal of tactile hyperalgesia.ConclusionThese results suggest that the presence of chemokine signaling by both injured and adjacent, uninjured sensory neurons is correlated with the maintenance phase of a persistent pain state, suggesting that chemokine receptor antagonists may be an important therapeutic intervention for chronic pain.

Nucleoside reverse transcriptase inhibitors (NRTIs) are known to produce painful neuropathies and to enhance states of pain hypersensitivity produced by HIV-1 infection. It has also been observed that in some neuropathic pain models, chemokines and their receptors are upregulated, perhaps contributing to the pain state. In order to understand if chemokines are involved in NRTI-mediated sensory neuropathies, we treated rats with the anti-retroviral drug, 2′,3′-dideoxycytidine (ddC), which is known to produce an extended period of hyperalgesia and allodynia. Using in situ hybridization, we observed that under normal conditions, CXCR4 chemokine receptors were widely expressed by satellite glia in the dorsal root ganglia (DRG) and Schwann cells in the sciatic nerve. A limited number of DRG neurons also expressed CXCR4 receptors. The chemokine SDF-1/ CXCL12 was similarly expressed in glial cells in the DRG and peripheral nerve. Following a single administration of ddC, expression levels of CXCR4 mRNA in glia and neurons and SDF-1 mRNA in glia increased considerably. The functional nature of increased CXCR4 mRNA expression was confirmed by measuring SDF-1 induced [Ca2+]i increases in acutely isolated DRG neurons and glia. In contrast, the expression of the chemokine receptors CCR2 and CCR5 did not change following ddC treatment. Pain hypersensitivity produced by ddC could be inhibited by treatment with the CXCR4 antagonist, AMD3100. Hence, we postulate that NRTIs produce pain hypersensitivity through the upregulation of CXCR4 signaling in the DRG. Increased numbers of CXCR4 receptors would also explain the synergism observed between NRTI treatment and the proalgesic effects of HIV-1 infection.

BackgroundMultiple adverse events are associated with the use of morphine for the treatment of chronic non-cancer pain, including opioid-induced hyperalgesia (OIH). Mechanisms of OIH are independent of opioid tolerance and may involve the morphine metabolite morphine-3-glucuronide (M3G). M3G exhibits limited affinity for opioid receptors and no analgesic effect. Previous reports suggest that M3G can act via the Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) heterodimer in the central nervous system to elicit pain.MethodsImmunoblot and immunocytochemistry methods were used to characterize the protein expression of TLR4 present in lumbar dorsal root ganglion (DRG). Using in vitro intracellular calcium and current clamp techniques, we determined whether TLR4 activation as elicited by the prototypical agonists of TLR4, lipopolysaccharide (LPS) and M3G, contributed to changes in intracellular calcium and increased excitation. Rodents were also injected with M3G to determine the degree to which M3G-induced tactile hyperalgesia could be diminished using either a small molecule inhibitor of the MD-2/TLR4 complex in rats or TLR4 knockout mice. Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 μm < DRG diameter <45 μm) for both control and M3G-treated neurons to determine the potential influence on voltage-gated sodium channels (NaVs).ResultsWe observed that TLR4 immunoreactivity was present in peptidergic and non-peptidergic sensory neurons in the DRG. Non-neuronal cells in the DRG lacked evidence of TLR4 expression. Approximately 15% of assayed small- and medium-diameter sensory neurons exhibited a change in intracellular calcium following LPS administration. Both nociceptive and non-nociceptive neurons were observed to respond, and approximately 40% of these cells were capsaicin-insensitive. Increased excitability observed in sensory neurons following LPS or M3G could be eliminated using Compound 15, a small molecule inhibitor of the TLR4/MD-2 complex. Likewise, systemic injection of M3G induced rapid tactile, but not thermal, nociceptive behavioral changes in the rat, which were prevented by pre-treating animals with Compound 15. Unlike TLR4 wild-type mice, TLR4 knockout mice did not exhibit M3G-induced hyperalgesia. As abnormal pain sensitivity is often associated with NaVs, we predicted that M3G acting via the MD-2/TLR4 complex may affect the density and gating of NaVs in sensory neurons. We show that M3G increases tetrodotoxin-sensitive and tetrodotoxin-resistant (NaV1.9) current densities.ConclusionsThese outcomes provide evidence that M3G may play a role in OIH via the TLR4/MD-2 heterodimer complex and biophysical properties of tetrodotoxin-sensitive and tetrodotoxin-resistant NaV currents.

BackgroundHigh-mobility group box-1 protein (HMGB1) is a nuclear protein that regulates gene expression throughout the body. It can also become cytoplasmic and function as a neuromodulatory cytokine after tissue damage or injury. The manner in which HMGB1 influences the peripheral nervous system following nerve injury is unclear. The present study investigated the degree to which HMGB1 signaling contributes to the maintenance of neuropathic pain behavior in the rodent.ResultsRedistribution of HMGB1 from the nucleus to the cytoplasm occurred in both sensory neurons derived from a tibial nerve injured (TNI) rat and in a sensory neuron-like cell line following exposure to a depolarizing stimulus. We also observe that exogenous administration of HMGB1 to acutely dissociated sensory neurons derived from naïve or TNI rodents elicit increased excitability. Furthermore systemic injection of glycyrrhizin (50 mg/kg; i.p.), a known inhibitor of HMGB1, reversed TNI-induced mechanical hyperalgesia at fourteen days and three months following nerve injury.ConclusionsWe have identified that a persistent endogenous release of HMGB1 by sensory neurons may be a potent, physiologically relevant modulator of neuronal excitability. More importantly, the use of the anti-inflammatory compound and known inhibitor of HMGB1, glycyrrhizin, has the ability to diminish persistent pain behavior in a model of peripheral neuropathy, presumably through its ability to neutralize the cyotkine. The identification of HMGB1 as a potential therapeutic target may contribute to a better understanding of mechanisms associated with chronic pain syndromes.

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na+-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na+ channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na+ channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7, a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na+ channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential.

Painful distal sensory polyneuropathy (DSP) is the most common neurological complication of HIV1 infection. Although infection with the virus itself is associated with an incidence of DSP, patients are more likely to become symptomatic following initiation of nucleoside reverse transcriptase inhibitor (NRTI) treatment. The chemokines monocyte chemoattractant protein-1 (MCP1/CCL2) and stromal derived factor-1 (SDF1/CXCL12) and their respective receptors, CCR2 and CXCR4, have been implicated in HIV1 related neuropathic pain mechanisms including NRTI treatment in rodents. Utilizing a rodent model that incorporates the viral coat protein, gp120, and the NRTI, 2'3'-dideoxycytidine (ddC), we examined the degree to which chemokine receptor signaling via CCR2 and CXCR4 potentially influences the resultant chronic hypernociceptive behavior. We observed that following unilateral gp120 sciatic nerve administration, rats developed profound tactile hypernociception in the hindpaw ipsilateral to gp120 treatment. Behavioral changes were also present in the hindpaw contralateral to the injury, albeit delayed and less robust. Using immunohistochemical studies, we demonstrated that MCP1 and CCR2 were upregulated by primary sensory neurons in lumbar ganglia by post-operative day (POD) 14. The functional nature of these observations was confirmed using calcium imaging in acutely dissociated lumbar dorsal root ganglion (DRG) derived from gp120 injured rats at POD 14. Tactile hypernociception in gp120 treated animals was reversed following treatment with a CCR2 receptor antagonist at POD 14. Some groups of animals were subjected to gp120 sciatic nerve injury in combination with an injection of ddC at POD 14. This injury paradigm produced pronounced bilateral tactile hypernociception from POD 14–48. More importantly, functional MCP1/CCR2 and SDF1/CXCR4 signaling was present in sensory neurons. In contrast to gp120 treatment alone, the hypernociceptive behavior associated with the injury plus drug combination was only effectively reversed using the CXCR4 antagonist AMD3100. These studies indicate that the functional upregulation of CCR2 and CXCR4 signaling systems following a combination of gp120 and an NRTI are likely to be of central importance to associated DSP and may serve as potential therapeutic targets for treatment of this syndrome.