Xavier Nadal

Crucial Role of CB2 Cannabinoid Receptor in the Regulation of Central Immune Responses during Neuropathic Pain

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB2 cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB2 knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB2 receptors. These results demonstrate the crucial role of CB2 cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB2 knock-outs, thus demonstrating the implication of the CB2 receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury

ABSTRACT Sigma‐1 receptor (σ1R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of σ1R in modulating nerve injury‐evoked pain. For this purpose, wild‐type mice and mice lacking the σ1R gene were exposed to partial sciatic nerve ligation and neuropathic pain‐related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal‐regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous σ1R knockout mice did not differ from wild‐type mice. Baseline values obtained in σ1R knockout mice before nerve injury in the plantar, cold‐plate and von Frey tests were also indistinguishable from those obtained in wild‐type mice. However, cold and mechanical allodynia did not develop in σ1R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking σ1R showed reduced wind‐up responses respect to wild‐type mice, as evidenced by a reduced number of action potentials induced by trains of C‐fiber intensity stimuli. In addition, in contrast to wild‐type mice, σ1R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind‐up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify σ1R as a constituent of the mechanisms modulating activity‐induced sensitization in pain pathways and point to σ1R as a new potential target for drugs designed to alleviate neuropathic pain.

Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia

&NA; Opioid receptors are major actors in pain control and are broadly distributed throughout the nervous system. A major challenge in pain research is the identification of key opioid receptor populations within nociceptive pathways, which control physiological and pathological pain. In particular, the respective contribution of peripheral vs. central receptors remains unclear, and it has not been addressed by genetic approaches. To investigate the contribution of peripheral delta opioid receptors in pain control, we created conditional knockout mice where delta receptors are deleted specifically in peripheral NaV1.8‐positive primary nociceptive neurons. Mutant mice showed normal pain responses to acute heat and to mechanical and formalin stimuli. In contrast, mutant animals showed a remarkable increase of mechanical allodynia under both inflammatory pain induced by complete Freund adjuvant and neuropathic pain induced by partial sciatic nerve ligation. In these 2 models, heat hyperalgesia was virtually unchanged. SNC80, a delta agonist administered either systemically (complete Freund adjuvant and sciatic nerve ligation) or into a paw (sciatic nerve ligation), reduced thermal hyperalgesia and mechanical allodynia in control mice. However, these analgesic effects were absent in conditional mutant mice. In conclusion, this study reveals the existence of delta opioid receptor‐mediated mechanisms, which operate at the level of NaV1.8‐positive nociceptive neurons. Delta receptors in these neurons tonically inhibit mechanical hypersensitivity in both inflammatory and neuropathic pain, and they are essential to mediate delta opioid analgesia under conditions of persistent pain. This delta receptor population represents a feasible therapeutic target to alleviate chronic pain while avoiding adverse central effects. The conditional knockout of delta‐opioid receptor in primary afferent NaV1.8 neurons augmented mechanical allodynia in persistent pain models and abolished delta opioid analgesia in these models.

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization

BACKGROUND AND PURPOSE The sigma‐1 (σ1) receptor is a ligand‐regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ1 receptor ligands used as pharmacological tools are unclear and the demonstration that σ1 receptor antagonists have efficacy in reversing central sensitization‐related pain sensitivity is still missing.

Interferon-γ Is a Critical Modulator of CB2 Cannabinoid Receptor Signaling during Neuropathic Pain

Nerve injuries often lead to neuropathic pain syndrome. The mechanisms contributing to this syndrome involve local inflammatory responses, activation of glia cells, and changes in the plasticity of neuronal nociceptive pathways. Cannabinoid CB2 receptors contribute to the local containment of neuropathic pain by modulating glial activation in response to nerve injury. Thus, neuropathic pain spreads in mice lacking CB2 receptors beyond the site of nerve injury. To further investigate the mechanisms leading to the enhanced manifestation of neuropathic pain, we have established expression profiles of spinal cord tissues from wild-type and CB2-deficient mice after nerve injury. An enhanced interferon-γ (IFN-γ) response was revealed in the absence of CB2 signaling. Immunofluorescence stainings demonstrated an IFN-γ production by astrocytes and neurons ispilateral to the nerve injury in wild-type animals. In contrast, CB2-deficient mice showed neuronal and astrocytic IFN-γ immunoreactivity also in the contralateral region, thus matching the pattern of nociceptive hypersensitivity in these animals. Experiments in BV-2 microglia cells revealed that transcriptional changes induced by IFN-γ in two key elements for neuropathic pain development, iNOS (inducible nitric oxide synthase) and CCR2, are modulated by CB2 receptor signaling. The most direct support for a functional involvement of IFN-γ as a mediator of CB2 signaling was obtained with a double knock-out mouse strain deficient in CB2 receptors and IFN-γ. These animals no longer show the enhanced manifestations of neuropathic pain observed in CB2 knock-outs. These data clearly demonstrate that the CB2 receptor-mediated control of neuropathic pain is IFN-γ dependent.

Neuropathic pain is enhanced in δ-opioid receptor knockout mice

We have evaluated the possible involvement of δ‐opioid receptor (DOR) in the development and expression of neuropathic pain. For this purpose, partial ligation of the sciatic nerve was performed in DOR knockout mice and wild‐type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold‐plate test and the plantar test, respectively. In wild‐type and DOR knockout mice, sciatic nerve injury led to a neuropathic pain syndrome revealed in these nociceptive behavioural tests. However, the development of mechanical and thermal allodynia, and thermal hyperalgesia was significantly enhanced in DOR knockout mice. These results reveal the involvement of DOR in the control of neuropathic pain and suggest a new potential therapeutic use of DOR agonists.

Pharmacological activation of 5-HT7 receptors reduces nerve injury-induced mechanical and thermal hypersensitivity

&NA; The involvement of the 5‐HT7 receptor in nociception and pain, particularly chronic pain (i.e., neuropathic pain), has been poorly investigated. In the present study, we examined whether the 5‐HT7 receptor participates in some modulatory control of nerve injury‐evoked mechanical hypersensitivity and thermal (heat) hyperalgesia in mice. Activation of 5‐HT7 receptors by systemic administration of the selective 5‐HT7 receptor agonist AS‐19 (1 and 10 mg/kg) exerted a clear‐cut reduction of mechanical and thermal hypersensitivities that were reversed by co‐administering the selective 5‐HT7 receptor antagonist SB‐258719. Interestingly, blocking of 5‐HT7 receptors with SB‐258719 (2.5 and 10 mg/kg) enhanced mechanical (but not thermal) hypersensitivity in nerve‐injured mice and induced mechanical hypersensitivity in sham‐operated mice. Effectiveness of the treatment with a 5‐HT7 receptor agonist was maintained after repeated systemic administration: no tolerance to the antiallodynic and antihyperalgesic effects was developed following treatment with the selective 5‐HT7 receptor agonist E‐57431 (10 mg/kg) twice daily for 11 days. The 5‐HT7 receptor co‐localized with GABAergic cells in the dorsal horn of the spinal cord, suggesting that the activation of spinal inhibitory GABAergic interneurons could contribute to the analgesic effects of 5‐HT7 receptor agonists. In addition, a significant increase of 5‐HT7 receptors was found by immunohistochemistry in the ipsilateral dorsal horn of the spinal cord after nerve injury, suggesting a “pain”‐triggered regulation of receptor expression. These results support the idea that the 5‐HT7 receptor subtype is involved in the control of pain and point to a new potential use of 5‐HT7 receptor agonists for the treatment of neuropathic pain.

A2A adenosine receptor regulates glia proliferation and pain after peripheral nerve injury

Abstract Peripheral nerve injury produces a persistent neuropathic pain state characterized by spontaneous pain, allodynia and hyperalgesia. In this study, we evaluated the possible involvement of A2ARs in the development of neuropathic pain and the expression of microglia and astrocytes in the spinal cord after sciatic nerve injury. For this purpose, partial ligation of the sciatic nerve was performed in A2A knockout mice and wild‐type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold‐plate test and the plantar test, respectively. In wild‐type animals, sciatic nerve injury led to a neuropathic pain syndrome that was revealed in these three nociceptive behavioural tests. However, a significant decrease of the mechanical allodynia and a suppression of thermal hyperalgesia and allodynia were observed in A2AR deficient mice. The expression of microglia and astrocytes was enhanced in wild‐type mice exposed to sciatic nerve injury and this response was attenuated in knockout animals. Taken together, our results demonstrate the involvement of A2ARs in the control of neuropathic pain and propose this receptor as an interesting target for the development of new drugs for the management of this clinical syndrome.

Involvement of the opioid and cannabinoid systems in pain control: new insights from knockout studies.

The endogenous opioid and cannabinoid systems are involved in the physiological inhibitory control of pain and are of particular interest for the development of therapeutic approaches for pain management. The involvement of these endogenous systems in pain control has been studied from decades by the use of compounds with different affinities for each cannabinoid and opioid receptor or for the different enzymes involved in endocannabinoid and endogenous opioid metabolism. However, the selectivity of these pharmacological tools in vivo has represented an important limitation for these studies. The generation of genetically modified mice with selective mutations in specific components of the endocannabinoid and endogenous opioid system has provided important advances in the identification of the specific contribution of each component of these endogenous systems in the perception of noxious stimuli and the development of pathological pain states. Different lines of constitutive and conditional knockout mice deficient in specific cannabinoid and opioid receptors, specific precursors of the endogenous opioid peptides and the main enzymes involved in endocannabinoid and endogenous opioid degradation are now available. These knockout mice have also been used to evaluate the contribution of each component of the endocannabinoid and opioid system in the antinociceptive effects of cannabinoid and opioid agonists, including those currently used to treat pain in humans. This review summarizes the main advances provided in the last 15 years by the use of these genetic tools in the knowledge of the physiological control of pain and the pharmacology of cannabinoid and opioid compounds for pain management.

Long‐lasting oral analgesic effects of N‐protected aminophosphinic dual ENKephalinase inhibitors (DENKIs) in peripherally controlled pain

The peripheral endogenous opioid system is critically involved in neuropathic and inflammatory pain generation as suggested by the modulation of opioid receptors expression and enkephalins (ENKs) release observed in these painful conditions. Accordingly, an innovative approach in the treatment of these nocifensive events is to increase and maintain high local concentrations of extracellular pain‐evoked ENKs, by preventing their physiological enzymatic inactivation by two Zn metallopeptidases, the neutral endopeptidase (NEP, neprilysin, EC 3.4.24.11) and the neutral aminopeptidase (APN, EC 3.4.11.2). With this aim, new orally active dual ENKephalinase inhibitors (DENKIs) were designed as soluble prodrugs by introducing a N‐terminal cleavable carbamate in the previously described aminophosphinic inhibitors. This induces long‐lasting antinociceptive responses after oral administration, in various rodent models of inflammatory and neuropathic pain. These responses are mediated through stimulation of peripheral opioid receptors by DENKIs‐protected ENKs as demonstrated by naloxone methiodide reversion. In all tested models, the most efficient prodrug 2a (PL265) was active, at least during 150–180 min, after single oral administration of 25–50 mg/kg in mice and of 100–200 mg/kg in rats. In models of neuropathic pain, both hyperalgesia and allodynia were markedly reduced. Interestingly, combination of inactive doses of 2a (PL265) and of the anti‐epileptic drug gabapentin had synergistic effect on neuropathic pain. Pharmacokinetic studies of 2a (PL265) in rats show that the active drug is the only generated metabolite produced. These encouraging results have made 2a (PL265) a suitable candidate for clinical development.