Marc-André Dansereau

CCL2 Released from Neuronal Synaptic Vesicles in the Spinal Cord Is a Major Mediator of Local Inflammation and Pain after Peripheral Nerve Injury

CCL2 chemokine and its receptor CCR2 may contribute to neuropathic pain development. We tested the hypothesis that injury to peripheral nerves triggers CCL2 release from afferents in the dorsal horn spinal cord (DHSC), leading to pronociceptive effects, involving the production of proinflammatory factors, in particular. Consistent with the release of CCL2 from primary afferents, electron microscopy showed the CCL2 immunoreactivity in glomerular boutons and secretory vesicles in the DHSC of naive rats. Through the ex vivo superfusion of DHSC slices, we demonstrated that the rate of CCL2 secretion was much lower in neonatal capsaicin-treated rats than in controls. Thus, much of the CCL2 released in the DHSC originates from nociceptive fibers bearing TRPV1 (transient receptor potential vanilloid 1). In contrast, high levels of CCL2 released from the DHSC were observed in neuropathic pain animal model induced by chronic constriction of the sciatic nerve (SN-CCI). The upregulated expression of proinflammatory markers and extracellular signal-regulated kinase (ERK) 1/2 pathway activation (ERK1/2 phosphorylation) in the DHSC of SN-CCI animals were reversed by intrathecal administration of the CCR2 antagonist INCB3344 (N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide). These pathological pain-associated changes in the DHSC were mimicked by the intrathecal injection of exogenous CCL2 in naive rats and were prevented by the administration of INCB3344 or ERK inhibitor (PD98059). Finally, mechanical allodynia, which was fully developed 2 weeks after SN-CCI in rats, was attenuated by the intrathecal injection of INCB3344. Our data demonstrate that CCL2 has the typical characteristics of a neuronal mediator involved in nociceptive signal processing and that antagonists of its receptor are promising agents from treating neuropathic pain.

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP‐1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP‐1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP‐1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co‐localized with pain‐related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium‐dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2‐induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.

Neurochemokines: a menage a trois providing new insights on the functions of chemokines in the central nervous system

J. Neurochem. (2011) 118, 680–694.

The Chemokine CCL2 Increases Nav1.8 Sodium Channel Activity in Primary Sensory Neurons through a Gβγ-Dependent Mechanism

Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Nav1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Nav1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Nav1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Nav1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Nav1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gβγ inhibitor), indicating the involvement of Gβγ released from PTX-sensitive Gi/o-proteins in the cross talk between CCR2 and Nav1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Nav1.8 currents via a CCR2/Gβγ-dependent mechanism.

Chemokine Network in the Nervous System: A New Target for Pain Relief

Physical insults including but not limited to nerve damage, inflammation, visceral pathologies and cancer generate long lasting pain commonly referred as chronic pain. Recently, members of the chemokine family and their receptors emerged as key modulators in nociceptive influx transmission in neuropathic and inflammatory chronic pain models. To this day, rodents defective in specific chemokine receptors have provided evidence of the implication of chemokine in pain sensitivity. In addition, up-regulation of chemokines and their receptors at multiple levels in the central nervous (CNS) and peripheral (PNS) systems is associated in the development of chronic pain. Indeed, we point out the fact that chemokines are synthesized and released by both neuronal and non-neuronal cells and act as neuromodulators. Even if their functional roles in the CNS remain largely unknown, chemokines participate in the glial activation and modulation of neuronal excitability as well as neurotransmitter release. This review focuses on three chemokines (i.e. CCL2, CXCL12, CX3CL1) recently identified as important mediators of the initiation and maintenance of pain hypersensitivity, thus broadening the panel of new strategies for the management of chronic pain.

Weight bearing evaluation in inflammatory, neuropathic and cancer chronic pain in freely moving rats

Preclinical pain assessment remains a key step for the development of new and potent painkillers. Significant progress in pain evaluation has been achieved with the development of non-reflexive tools. Seeking efficient and clinically relevant devices for pain-related quality of life assessment, we evaluated a new Dynamic Weight Bearing (DWB) device based on pressure captors in three different preclinical chronic pain models. Inflammatory (CFA), neuropathic (CCI) and bone cancer pain (femoral tumor) models were evaluated in Sprague Dawley rats for mechanical allodynia using dynamic von Frey for pain-related behaviors and DWB for discomfort. We observed similar impairment patterns in all of the models for both von Frey (allodynia) and DWB (weight balance) during the complete observation period, starting at day 3 in CCI- and CFA-affected limbs and at day 14 in bone cancer-afflicted rats, indicating that the DWB could be a useful tool for supporting pain assessment. Interestingly, we demonstrated that the main compensation, when animals experienced pain, was seen in the forepaws, ranging from 46% to 69% of increased load compared to normal. Other pain-related coping behaviors were also measured, such as the time spent on each paw and the contact surface. Our results revealed that CFA, CCI and cancerous rats decreased the use of their ipsilateral hind paws by 30% and showed a 50% reduction in paw surface pressed against the floor. In conclusion, this new device improves methods for preclinical evaluation of discomfort and quality of life proxies and could be helpful in screening putative analgesics.

Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.

Functional up-regulation of Nav1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

BackgroundFunctional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Nav1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Nav1.8 in controlling Aβ-fiber excitability following persistent inflammation.MethodsDistribution and expression of Nav1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund’s adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Nav1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Nav1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats.ResultsOur findings revealed that Nav1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Nav1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Nav1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Nav1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Nav1.8 regulation in Aβ-fibers contributes to inflammatory pain.ConclusionsCollectively, these findings support a key role for Nav1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.

Functional inhibition of chemokine receptor CCR2 by dicer-substrate-siRNA prevents pain development

Background Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund’s adjuvant. Results To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2′-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund’s adjuvant-induced inflammatory chronic pain model. Conclusion Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain.