Emer M. Garry

Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain.

BACKGROUND Chronic established pain, especially that following nerve injury, is difficult to treat and represents a largely unmet therapeutic need. New insights are urgently required, and we reasoned that endogenous processes such as cooling-induced analgesia may point the way to novel strategies for intervention. Molecular receptors for cooling have been identified in sensory nerves, and we demonstrate here how activation of one of these, TRPM8, produces profound, mechanistically novel analgesia in chronic pain states. RESULTS We show that activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of specific pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, thereby inhibiting the characteristic sensitization of dorsal-horn neurons and behavioral-reflex facilitation. TRPM8 expression was increased in a subset of sensory neurons after nerve injury. The essential role of TRPM8 in suppression of sensitized pain responses was corroborated by specific knockdown of its expression after intrathecal application of an antisense oligonucleotide. We further show that the analgesic effect of TRPM8 activation is centrally mediated and relies on Group II/III metabotropic glutamate receptors (mGluRs), but not opioid receptors. We propose a scheme in which Group II/III mGluRs would respond to glutamate released from TRPM8-containing afferents to exert an inhibitory gate control over nociceptive inputs. CONCLUSIONS TRPM8 and its central downstream mediators, as elements of endogenous-cooling-induced analgesia, represent a novel analgesic axis that can be exploited in chronic sensitized pain states.

Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves

Nerve impulses are propagated at nodes of Ranvier in the myelinated nerves of vertebrates. Internodal distances have been proposed to affect the velocity of nerve impulse conduction; however, direct evidence is lacking, and the cellular mechanisms that might regulate the length of the myelinated segments are unknown. Ramón y Cajal described longitudinal and transverse bands of cytoplasm or trabeculae in internodal Schwann cells and suggested that they had a nutritive function. Here we show that internodal growth in wild-type nerves is precisely matched to nerve extension, but disruption of the cytoplasmic bands in Periaxin-null mice impairs Schwann cell elongation during nerve growth. By contrast, myelination proceeds normally. The capacity of wild-type and mutant Schwann cells to elongate is cell-autonomous, indicating that passive stretching can account for the lengthening of the internode during limb growth. As predicted on theoretical grounds, decreased internodal distances strikingly decrease conduction velocities and so affect motor function. We propose that microtubule-based transport in the longitudinal bands of Cajal permits internodal Schwann cells to lengthen in response to axonal growth, thus ensuring rapid nerve impulse transmission.

Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice

The Prx gene in Schwann cells encodes L- and S-periaxin, two abundant PDZ domain proteins thought to have a role in the stabilization of myelin in the peripheral nervous system (PNS). Mice lacking a functional Prx gene assemble compact PNS myelin. However, the sheath is unstable, leading to demyelination and reflex behaviors that are associated with the painful conditions caused by peripheral nerve damage. Older Prx-/- animals display extensive peripheral demyelination and a severe clinical phenotype with mechanical allodynia and thermal hyperalgesia, which can be reversed by intrathecal administration of a selective NMDA receptor antagonist We conclude that the periaxins play an essential role in stabilizing the Schwann cell-axon unit and that the periaxin-deficient mouse will be an important model for studying neuropathic pain in late onset demyelinating disease.

Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs

&NA; Reactivation of latent varicella zoster virus (VZV) within sensory trigeminal and dorsal root ganglia (DRG) neurons produces shingles (zoster), often accompanied by a chronic neuropathic pain state, post‐herpetic neuralgia (PHN). PHN persists despite latency of the virus within human sensory ganglia and is often unresponsive to current analgesic or antiviral agents. To study the basis of varicella zoster‐induced pain, we have utilised a recently developed model of chronic VZV infection in rodents. Immunohistochemical analysis of DRG following VZV infection showed the presence of a viral immediate early gene protein (IE62) co‐expressed with markers of A‐ (neurofilament‐200; NF‐200) and C‐ (peripherin) afferent sensory neurons. There was increased expression of neuropeptide Y (NPY) in neurons co‐expressing NF‐200. In addition, there was an increased expression of α2δ1 calcium channel, Nav1.3 and Nav1.8 sodium channels, the neuropeptide galanin and the nerve injury marker, Activating Transcription Factor‐3 (ATF‐3) as determined by Western blotting in DRG of VZV‐infected rats. VZV infection induced increased behavioral reflex responsiveness to both noxious thermal and mechanical stimuli ipsilateral to injection (lasting up to 10 weeks post‐infection) that is mediated by spinal NMDA receptors. These changes were reversed by systemic administration of gabapentin or the sodium channel blockers, mexiletine and lamotrigine, but not by the non‐steroidal anti‐inflammatory agent, diclofenac. This is the first time that the profile of VZV infection‐induced phenotypic changes in DRG has been shown in rodents and reveals that this profile appears to be broadly similar (but not identical) to changes in other neuropathic pain models.

Specific involvement in neuropathic pain of AMPA receptors and adapter proteins for the GluR2 subunit

Chronic pain states arise from peripheral nerve injury and are inadequately treated with current analgesics. Using intrathecal drug administration in a rat model of neuropathic pain, we demonstrate that AMPA receptors play a role in the central sensitisation that is thought to underpin chronic pain. The GluR2 subunit of the AMPA receptor binds to a number of intracellular adapter proteins including GRIP, PICK1 and NSF, which may link the receptor to proteins with signalling, scaffolding and other roles. We implicate for the first time a possible role for GRIP, PICK1 and NSF in neuropathic sensitisation from experiments with cell-permeable blocking peptides mimicking their GluR2 interaction motifs and also demonstrate differential changes in expression of these proteins following peripheral nerve injury. These studies suggest a critical involvement of protein:protein complexes associated with the AMPA receptor in neuropathic pain, and the possibility that they may have potential as novel therapeutic targets.

NMDA receptor antagonist treatment at the time of nerve injury prevents injury-induced changes in spinal NR1 and NR2B subunit expression and increases the sensitivity of residual pain behaviours to subsequently administered NMDA receptor antagonists

&NA; Spinal NMDA receptors (NMDA R) are important in neuropathic sensitisation and acute administration of antagonists can provide temporary attenuation of sensitisation. If establishment of the chronic pain state could be prevented by brief administration of such agents at or around the time of nerve injury (pre‐emptive analgesia) it might be possible to avoid many of the unacceptable side effects associated with repeated administration of these or other antagonists. Several reports describe aspects of effective pre‐emptive analgesia from NMDA R antagonists in animal models of neuropathic pain. The first aim of the present study was to make a direct comparison of changes in mechanical allodynia, cold allodynia and thermal hyperalgesia following nerve injury, demonstrating their increasing degree of susceptibility to pre‐emptive NMDA R antagonist treatment. Secondly, we used immunoblotting and immunohistochemistry to investigate the effects of nerve injury on NMDA receptor subunit expression, revealing increased expression of NR2B, but not NR2A and reduced NR1 in the superficial dorsal horn. These changes were attenuated following NMDA receptor antagonist pre‐treatment. Thirdly, we investigated the pharmacological properties of residual mechanical allodynia and cold allodynia that remained after pre‐emptive treatment and revealed a greater sensitivity to NMDA R antagonists. These findings indicate that in addition to a marked suppression of thermal hyperalgesia and cold allodynia, pre‐emptive treatment with NMDA R antagonist causes a lasting change in spinal NMDA R complexes such that remaining mechanical allodynia should be more effectively targeted by NMDA R antagonists.

Activation of p38 and p42/44 MAP kinase in neuropathic pain: involvement of VPAC2 and NK2 receptors and mediation by spinal glia.

Activation of intracellular signaling pathways involving p38 and p42/44 MAP kinases may contribute importantly to synaptic plasticity underlying spinal neuronal sensitization. Inhibitors of p38 or p42/44 pathways moderately attenuated responses of dorsal horn neurons evoked by mustard oil but not brush and alleviated the behavioral reflex sensitization seen following nerve injury. Activation of p38 and p42/44 MAP kinases in spinal cord ipsilateral to constriction injury was reduced by antagonists of NMDA, VPAC2 and NK2 (but not related) receptors, the glial inhibitor propentofylline and inhibitors of TNF-alpha. A VPAC2 receptor agonist enhanced p38 phosphorylation and caused behavioral reflex sensitization in naïve animals that could be blocked by co-administration of p38 inhibitor. Conversely, an NK2 receptor agonist activated p42/44 and caused behavioral sensitization that could be prevented by co-administration of p42/44 inhibitor. Thus, spinal p38 and p42/44 MAP kinases are activated in neuropathic pain states by mechanisms involving VPAC2, NK2, NMDA receptors and glial cytokine production.

A new view on how AMPA receptors and their interacting proteins mediate neuropathic pain.

The sensitised somatosensory responses of neuropathic pain are resistant to conventional analgesia. Hence, it is important to reveal new targets for the design of therapeutic agents. Evidence generated over the last thirty years has established AMPA and NMDA glutamate receptor subtypes as participants in the development and maintenance of chronic pain states (Dickenson and Sullivan, 1987). These receptors are expressed at high levels at the first sensory synaptic relays in the spinal cord. Importantly, it has become clear that spinal mechanisms underlying neuropathic sensitisation appear to represent a distinct form of neural plasticity in the central nervous system. Neuropathic sensitisation results in an increase in the excitability of nociceptive neurons in laminae I-II of the superficial dorsal horn and heightened pain perception. Neuropathic sensitisation is a heterosynaptic facilitation brought about by relatively low frequency activation of diverse inputs, which is quite different from high frequency long-term potentiation (LTP) (Ji et al., 2003). Although LTP-like events can be observed in spinal cord slices following much higher frequency stimulation, they cannot be detected in intact spinal cord, where supraspinal and other modulatory influences are present (Sandkühler and Liu, 1998). Both of these central mechanisms of plasticity share dependence on the NMDA and AMPA subtypes of glutamate receptor. The therapeutic potential of AMPA receptor blockers in chronic sensitised pain states has been largely overlooked, because AMPA receptors contribute to the acute spinal processing of both nociceptive and nonnociceptive inputs in the spinal cord (Garry et al., 2003). The intrathecal application of a number of AMPA receptor blockers (including agents with very high selectivity) causes marked reversal of the sensitised thermal hyperalgesia and mechanical allodynia, at doses that do not affect unsensitised or naı̈ve responses or motor function (Garry et al., 2003). These findings are consistent with reports of a role for both NMDA and AMPA receptors in neuropathic hyperalgesia (Mao et al., 1992) and of an allodynic state induced by spinal administration of AMPA plus metabotropic glutamate receptor (mGluR) 1/5 agonists (Meller et al., 1996). Pharmacological disruption of AMPA receptors may therefore represent a useful new strategy for the treatment of neuropathic pain. In particular, GluR2-deficient AMPA receptor complexes are implicated in neuropathic sensitisation, since Joro Spider Toxin (JSTx), a selective blocker of GluR2-deficient complexes, attenuates secondary mechanical allodynia (Sorkin et al., 2001). JSTx also modulates C-afferentinduced acute responses and the ‘wind-up’ of dorsal horn neurons that occurs following repetitive C-fiber stimulation (Stanfa et al., 2000). A number of proteins that interact with the intracellular C-termini of postsynaptic AMPA receptor GluR1-4 subunits have been identified (Hirbec et al., 2002). Receptor localisation and the ability to regulate intracellular signal transduction machinery may be crucially determined by these interactions. Indeed, increasing evidence indicates that regulated synaptic insertion of AMPA receptors, through the interactions of subunits with protein partners, can play a key role in increasing synaptic efficiency (Schnell et al., 2002). This raises the question of whether selective disruption of AMPA receptor subunit interactions with their partner proteins could represent a novel route for the design of refined therapeutic agents (Fig. 1). Pain 109 (2004) 210–213 www.elsevier.com/locate/pain

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino‐acid substitution in the polyproline‐binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol‐3‐kinase‐C2α to the SH3‐binding site of PSD95. In wild‐type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.