Fabien Marchand

Role of the Immune system in chronic pain

During the past two decades, an important focus of pain research has been the study of chronic pain mechanisms, particularly the processes that lead to the abnormal sensitivity — spontaneous pain and hyperalgesia — that is associated with these states. For some time it has been recognized that inflammatory mediators released from immune cells can contribute to these persistent pain states. However, it has only recently become clear that immune cell products might have a crucial role not just in inflammatory pain, but also in neuropathic pain caused by damage to peripheral nerves or to the CNS.

Immune and glial cell factors as pain mediators and modulators

A decade ago the attention of pain scientists was focused on a small number of molecules such as prostaglandin and bradykinin as peripheral pain mediators or modulators. These factors were known to be produced by tissue damage or inflammation, and considered responsible for the activation and sensitization of peripheral pain signaling sensory neurons. A small number of molecules were also identified as central pain mediators, most notably glutamate and substance P released from central nociceptive nerve terminals, and, starting at that time, appreciation that nitric oxide might be produced by dorsal horn neurons and act as a diffusible transmitter to increase excitability of central pain circuits. During the last decade evidence has emerged for many novel pain mediators. The old ones have not disappeared, although their roles have been redefined in some cases. Prostaglandin E2 (PGE2), for instance, is now recognized as playing a prominent role in CNS as well as peripheral tissues. The newly identified mediators include a variety of factors produced and released from nonneuronal cells-predominantly immune and glial cells. The evidence is now growing apace that these are important mediators of persistent pain states and can act at a number of loci. Here we review the actions of several of these factors-the pro-inflammatory cytokines, some chemokines, and some neurotrophic factors, which, in addition to their traditionally recognized roles, are all capable of changing the response properties of peripheral and central pain signaling neurons. We review these actions, first in periphery, where a substantial literature has accumulated, and then in spinal cord, where the role of factors from nonneuronal cells has only recently been identified as of considerable importance.

Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain

A recent major conceptual advance has been the recognition of the importance of immune system–neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS-induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.

Pathophysiology of peripheral neuropathic pain: immune cells and molecules.

Damage to the peripheral nervous system often leads to chronic neuropathic pain characterized by spontaneous pain and an exaggerated response to painful and/or innocuous stimuli. This pain condition is extremely debilitating and usually difficult to treat. Although inflammatory and neuropathic pain syndromes are often considered distinct entities, emerging evidence belies this strict dichotomy. Inflammation is a well-characterized phenomenon, which involves a cascade of different immune cell types, such as mast cells, neutrophils, macrophages, and T lymphocytes. In addition, these cells release numerous compounds that contribute to pain. Recent evidence suggests that immune cells play a role in neuropathic pain in the periphery. In this review we identify the different immune cell types that contribute to neuropathic pain in the periphery and release factors that are crucial in this particular condition.

CCL2 is a key mediator of microglia activation in neuropathic pain states

While neuroimmune interactions are increasingly recognized as important in nociceptive processing, the nature and functional significance of these interactions is not well defined. There are multiple reports that the activation of spinal microglia is a critical event in the generation of neuropathic pain behaviors but the mediators of this activation remain disputed. Here we show that the chemokine CCL2, produced by both damaged and undamaged primary sensory neurons in neuropathic pain states in rats, is released in an activity dependent manner from the central terminals of these fibres. We also demonstrate that intraspinal CCL2 in naïve rats leads to activation of spinal microglia and neuropathic pain‐like behavior. An essential role for spinal CCL2 is demonstrated by the inhibition of neuropathic pain behavior and microglial activation by a specific neutralising antibody to CCL2 administered intrathecally. Thus, the neuronal expression of CCL2 provides a mechanism for immune activation, which in turn regulates the sensitivity of pain signaling systems in neuropathic pain states.

P2X7-Dependent Release of Interleukin-1β and Nociception in the Spinal Cord following Lipopolysaccharide

The cytokine interleukin-1β (IL-1β) released by spinal microglia in enhanced response states contributes significantly to neuronal mechanisms of chronic pain. Here we examine the involvement of the purinergic P2X7 receptor in the release of IL-1β following activation of Toll-like receptor-4 (TLR4) in the dorsal horn, which is associated with nociceptive behavior and microglial activation. We observed that lipopolysaccharide (LPS)-induced release of IL-1β was prevented by pharmacological inhibition of the P2X7 receptor with A-438079, and was absent in spinal cord slices taken from P2X7 knock-out mice. Application of ATP did not evoke release of IL-1β from the dorsal horn unless preceded by an LPS priming stimulus, and this release was dependent on P2X7 receptor activation. Extensive phosphorylation of p38 MAPK in microglial cells in the dorsal horn was found to correlate with IL-1β secretion following both LPS and ATP. In behavioral studies, intrathecal injection of LPS in the lumbar spinal cord produced mechanical hyperalgesia in rat hindpaws, which was attenuated by concomitant injections of either a nonspecific (oxidized ATP) or a specific (A-438079) P2X7 antagonist. In addition, LPS-induced hypersensitivity was observed in wild-type but not P2X7 knock-out mice. These data suggest a critical role for the P2X7 receptor in the enhanced nociceptive transmission associated with microglial activation and secretion of IL-1β in the dorsal horn. We suggest that CNS-penetrant P2X7 receptor antagonists, by targeting microglia in pain-enhanced response states, may be beneficial for the treatment of persistent pain.

Effects of Etanercept and Minocycline in a rat model of spinal cord injury

Loss of function is usually considered the major consequence of spinal cord injury (SCI). However, pain severely compromises the quality of life in nearly 70% of SCI patients. The principal aim of this study was to assess the contribution of Tumor necrosis factor α (TNF‐α) to SCI pain. TNF‐α blockers have already been successfully used to treat inflammatory disorders but there are few studies on its effect on neuropathic pain, especially following SCI. Following T13 spinal cord hemisection, we examined the effects on mechanical allodynia and microglial activation of immediate and delayed chronic intrathecal treatment with etanercept, a fusion protein blocker of TNF‐α. Immediate treatment (starting at the time of injury) with etanercept resulted in markedly reduced mechanical allodynia 1, 2, 3 and 4 weeks after SCI. Delayed treatment had no effect. Immediate etanercept treatment also reduced spinal microglial activation assessed by OX‐42 immunostaining, a putative marker of activated microglia. To assess whether the effects of etanercept were mediated via decreased microglial activation, we examined the effects of the microglial inhibitor, minocycline which significantly reduced the development of pain behaviours at 1 and 2 weeks after SCI compared to saline treatment. Minocycline also significantly reduced microglial OX‐42 expression. Furthermore, minocycline decreased the expression of noxious‐stimulation‐induced c‐Fos, suggesting an effect on evoked neuronal activity. This study demonstrates that TNF‐α plays an important role in the establishment of neuropathic pain following SCI, seemingly dependent on microglial activation. Pharmacological targeting of TNF‐α may offer therapeutic opportunities for treating SCI pain.

Phosphatidylinositol 3-Kinase Is a Key Mediator of Central Sensitization in Painful Inflammatory Conditions

Here, we show that phosphatidylinositol 3-kinase (PI3K) is a key player in the establishment of central sensitization, the spinal cord phenomenon associated with persistent afferent inputs and contributing to chronic pain states. We demonstrated electrophysiologically that PI3K is required for the full expression of spinal neuronal wind-up. In an inflammatory pain model, intrathecal administration of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent PI3K inhibitor, dose-dependently inhibited pain-related behavior. This effect was correlated with a reduction of the phosphorylation of ERK (extracellular signal-regulated kinase) and CaMKII (calcium/calmodulin-dependent protein kinase II). In addition, we observed a significant decrease in the phosphorylation of the NMDA receptor subunit NR2B, decreased translocation to the plasma membrane of the GluR1 (glutamate receptor 1) AMPA receptor subunit in the spinal cord, and a reduction of evoked neuronal activity as measured using c-Fos immunohistochemistry. Our study suggests that PI3K is a major factor in the expression of central sensitization after noxious inflammatory stimuli.

Gabapentin reverses microglial activation in the spinal cord of streptozotocin‐induced diabetic rats

Diabetes mellitus is the leading cause of peripheral neuropathy worldwide. Despite this high level of incidence, underlying mechanisms of the development and maintenance of neuropathic pain are still poorly understood. Evidence supports a prominent role of glial cells in neuropathic pain states. Gabapentin is used clinically and shows some efficacy in the treatment of neuropathic pain. Here we investigate the distribution and activation of spinal microglia and astrocytes in streptozotocin (STZ)‐diabetic rats and the effect of the gold standard analgesic, Gabapentin, on these cells. Mechanical allodynia was observed in four week‐diabetic rats. Oral administration of Gabapentin significantly attenuated mechanical allodynia. Quantification of cell markers Iba‐1 for microglia and GFAP for astrocytes revealed extensive activation of microglia in the dorsal horn of diabetic rats, whereas a reduction in the number of astrocytes could be observed. In addition, an attenuation of microglial activation correlated with reduced allodynia following Gabapentin treatment, while Gabapentin had no effect on the number of astrocytes. Here we show a role of microglia in STZ‐induced mechanical allodynia and furthermore, that the anti‐allodynic effect of Gabapentin may be linked to a reduction of spinal microglial activation. Astrocytic activation in this model appears to be limited and is unaffected by Gabapentin treatment. Consequently, spinal microglial activation is a key mechanism underlying diabetic neuropathy. Furthermore, we suggest that Gabapentin may exert its anti‐allodynic actions partially through alterations of microglial cell function.

Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats

Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.