In both cases a combination of mechanisms results in augmented nociceptive transmission due to peripheral and central sensitisation. Neuropathic pain is that arising from a lesion to the PNS (peripheral nervous system) or CNS as a consequence of physical trauma or disease pathogenesis. Chronic inflammatory pain is typically associated with inflammatory diseases such as arthritis, where the presence of algogenic mediators results in nociceptor sensitisation in the affected tissue, such as the joints in the case of arthritis. Chronic pain is characterised by the presence of a range of symptoms including hyperalgesia (increased sensitivity to noxious stimuli), allodnyia (a painful response to a previously innocuous stimuli) and spontaneous pain (Woolf and Mannion 1999). The treatment of chronic pain is a substantial problem within the clinic as many patients do not respond adequately to available analgesics. The difficulty in treating chronic pain conditions is thought to be a consequence of the heterogeneity of the molecular mechanisms underlying the development and maintenance of chronic pain, many of which are not well understood.

The mechanisms by which these maladaptive pain states arise can be categorised into peripheral sensitisation (changes in peripheral nerves) and central sensitisation (including immune responses in the spinal cord). Peripheral sensitisation due to injury to a localised area leads to primary hyperalgesia and results in increased nociceptive transmission from the periphery which causes changes in dorsal horn neuron activation. Dorsal horn neurons exhibit reduced thresholds to noxious stimuli applied to the periphery, de novo excitation by previously innocuous stimuli, expansion of their receptive fields and increased spontaneous activity. These changes increase excitability of CNS neurons and result in central sensitisation, which is fundamental for the generation of allodynia, secondary hyperalgesia and spontaneous pain (Kuner 2010; Latremoliere and Woolf 2009; Sandkuhler 2009). The resulting increase in glutamate released from central terminals of sensitised neurons trigger phosphorylation of NMDA receptors and post-translational modifications of neuronal proteins. Glial cells within the spinal cord respond to the enhanced nociceptive input by proliferating and switching to a responsive state whereby they release mediators which contribute to dorsal horn mechanisms of chronic pain (Clark and Malcangio 2012; Old and Malcangio 2012). Enhanced activity within the spinal cord triggers cortical and subcortical structures to facilitate excitation and signalling and constitutes higher centre modulation (D’Mello and Dickenson 2008).

Here we will describe and define some of the key mechanisms by which microglia and astrocyte responses in the dorsal horn contribute to the development and maintenance of chronic neuropathic and inflammatory pain states, with a specific focus on chemokine/cytokine signalling and second messenger activation.

Microglia are the resident immune cells of the CNS and as such respond to pathological insult or tissue injury and subsequent release of mediators from damaged cells. These cells respond to a number of mediators that are increased in the dorsal horn following peripheral injury. They express receptors for the neurotransmitters released from the central terminals of primary afferents: glutamate NMDA and AMPA receptors, the SP receptor NK1 and the CGRP receptor, TrkB receptors for brain derived neurotrophic factor (BDNF) and purinergic receptors including P2X7 and P2X4, many of which are upregulated following injury to the periphery (Rasley et al. 2002; Pezet et al. 2002; McMahon and Malcangio 2009; Ransohoff and Perry 2009).

Microglia respond quickly to an insult by proliferating to expand their population; this process is often termed microgliosis. An insult need not be central in nature, for example it could be the result of a CNS infection or spinal cord injury. In rodent peripheral nerve injury models of neuropathic pain, microglia within the dorsal horn of the spinal cord respond swiftly to augmented primary afferent fibre input. This phenotypic shift is often referred to as the ‘activation’ of microglia. Input from the periphery is vital for this process; blockade of peripheral nerve conduction by the application of a local anaesthetic prevents nerve injury-induced microglial response (Wen et al. 2007; Hathway et al. 2009; Suter et al. 2009). In the region of the dorsal horn in which the injured primary afferents terminate, microglia increase in their numbers and appear more amoeboid, with a hypertrophied soma and thick retracted processes. Additionally they alter their expression of cell surface antigens that play a critical role in immune responses; for example, MHC class II is increased allowing the presentation of antigens by microglia (McMahon and Malcangio 2009). Activated microglia also synthesise and release a variety of pro-nociceptive mediators into the extracellular environment, including cytokines (e.g. TNF and IL-1β), chemokines (e.g. CCL2), reactive oxygen species (ROS) and nitric oxide (NO). Prominent signalling pathways in the development of neuropathic pain are the CX3CR1/L1 loop (discussed below) and the purinergic pathway of microglial activation and subsequent BDNF release. ATP is known to stimulate microglia both in vivo and in vitro via purinergic receptors on the surface membrane of the cells (Honda et al. 2001; Tsuda et al. 2003, 2012; Davalos et al. 2005). Microglial P2X4 is of particular importance for the development of neuropathic pain; it is upregulated in microglia as early as 24 hours after peripheral nerve injury, and the pharmacological inhibition of this receptor transiently attenuates mechanical allodynia in rodents. Furthermore, the intrathecal administration of ATP-treated microglia in naive mice results in the development of mechanical hypersensitivity (Tsuda et al. 2003; Coull et al. 2005). Current evidence indicated that downstream effects of the activation of microglial purinergic receptors are mediated by the release of BDNF via the phosphorylation of p38 MAPK; the administration of an inhibitor of the BDNF receptor, TrkB, prevents the behavioural changes observed following the administration of activated microglia (Coull et al. 2005), and a p38 MAPK inhibitor is able to prevent release of BDNF from microglia (Trang et al. 2009).

The temporal profile of microglial activation within the spinal cord is well documented in models of neuropathic pain. Real-time PCR of spinal cord tissue extracts demonstrates alterations in the levels of microglia-specific mRNA transcripts (e.g. TLR4 and CD14) within hours of injury (Tanga et al. 2004), and the phosphorylation of the microglial MAPK p38 is evident within minutes from peripheral nerve injury (Svensson et al. 2005b; Clark et al. 2007b). Morphological changes (the presence of amoeboid cells and increased immunoreactivity for the microglial marker Ox42) have been observed immunohistochemically as early as 3 days after nerve injury and are maintained for at least 50 days post-surgery (Clark et al. 2007a). Furthermore, the temporal profile of microglial activation is concomitant to the development of behavioural hypersensitivity (Clark et al. 2007b; Zhang et al. 2007; Peters et al. 2007) and the intrathecal administration of compounds that inhibit glial activation to neuropathic rodents is able to attenuate pain behaviours (Clark et al. 2007a; Tawfik et al. 2007), demonstrating that the activation of these cells does indeed contribute to aberrant pain signalling.

Due to their many functions in the maintenance of CNS homeostasis, under physiological conditions astrocytes are often referred to as ‘active’. Following a change in their environment, such as that occurring in the spinal cord in models of chronic pain, these cells shift to a ‘reactive’ phenotype; astrocytes undergo hypertrophy and increase their expression of a number of cellular proteins including GFAP, S100β and vimentin, which are consequently often used as markers of astrocyte reactivity (Ridet et al. 1997; Pekny and Nilsson 2005). This process is referred to as astrogliosis and has been demonstrated in a number of surgical models of neuropathic pain (Garrison et al. 1991; Colburn et al. 1999; Sweitzer et al. 1999). Astrocyte reactivity can occur through the activation of several pathways; these cells express the receptors for the neurotransmitters NMDA, SP and CGRP (Porter and McCarthy 1997) and thus respond to increased transmitter release in the dorsal horn of the spinal cord subsequent to peripheral nerve injury. In addition, astrocytes possess a variety of cytokine and chemokine receptors that can be activated by their ligands released from microglia; key examples include IL-18/IL-18R, TNF/TNFR and CCL2/CCR2 (Miyoshi et al. 2008; Gao et al. 2009, 2010b). One of the astrocytic changes most likely to be responsible for the contribution of these cells to the maintenance of pain is a decrease in the glutamate transporters GLT1 and GLAST. These transporters function to regulate extracellular glutamate concentrations at non-toxic levels. A decrease in their expression or function results in an elevation in the concentration of spinal extracellular glutamate which can elicit nociceptive hypersensitivity via the activation of NMDA and AMPA receptors (Liaw et al. 2005; Weng et al. 2006). Recent evidence indicates that astrocytes, like microglia, play a critical role in aberrant pain signalling, as the administration of glial inhibitors, such as fluorocitrate, attenuates pain behaviours in rodents (Milligan et al. 2003; Watkins et al. 1997; Okada-Ogawa et al. 2009). Interestingly, the intrathecal administration of reactive astrocytes, briefly incubated with TNF, induces the development of mechanical allodynia in uninjured mice (Gao et al. 2010b). As with microglia, the temporal profile of astrocyte activation has been assessed; astrogliosis becomes apparent after the microglial response, several days after peripheral nerve injury, and lasts for over 4 weeks post-injury (Colburn et al. 1999; Tanga et al. 2004; Romero-Sandoval et al. 2008). Additionally genetically altered mice that are deficient in GFAP develop a shorter lasting mechanical allodynia than their wild-type counterparts. Finally the administration of a GFAP antisense mRNA that prevents translation of GFAP reverses established mechanical allodynia when administered to rats 6 weeks following a peripheral nerve injury (Kim et al. 2009). Together these data demonstrate a role for astrocytes in the maintenance of neuropathic pain.

CX3CL1, also known as fractalkine, is the only member of the CX3C family of cytokines. The cx3cl1 gene was originally described as abundant in the brain and heart but present in all tissues assessed, except peripheral blood leukocytes (Pan et al. 1997). Recent evidence obtained from a series of in situ hybridisation and immunohistochemical studies has determined that neurons are the principle source of CX3CL1 in the CNS (Hughes et al. 2002; Nishiyori et al. 1998; Tarozzo et al. 2003). Additionally, the development of a transgenic mouse expressing a red fluorescent reporter under the control of the CX3CL1 promoter has demonstrated that within the spinal cord CX3CL1 is expressed in dorsal horn neurons (Kim et al. 2011). Both the CX3CL1 protein and mRNA are expressed here, but the protein is not upregulated following peripheral nerve injury (Verge et al. 2004; Clark et al. 2009; Lindia et al. 2005). Furthermore, it has been demonstrated that cytoplasmic glutamate-containing vesicles are present within CX3CL1-positive neurons, indicating that CX3CL1 is expressed within excitatory neurons (Tong et al. 2000).

First described as a potent chemoattractant of T-cells and monocytes in the late 1990s (Bazan et al. 1997; Pan et al. 1997), CX3CL1 exists in two forms. As a membrane-tethered protein it plays a critical role in the firm adhesion of leukocytes to the endothelium to facilitate transmigration (Imai et al. 1997; Fong et al. 1998; Corcione et al. 2009). Soluble CX3CL1 is produced by metalloproteases (ADAM10/17) or protease (Cathepsin S) -mediated cleavage of membrane-bound CX3CL1. Whilst ADAM 10 is responsible for constitutive shedding of CX3CL1, ADAM 17 facilitates inducible shedding of this protein (Bazan et al. 1997; Hundhausen et al. 2003, 2007; Clark et al. 2011). Cathepsin S, on the other hand, is expressed by antigen-presenting cells such as microglia where its release is dependent on the activation of the purinergic receptor P2X7 (Clark et al. 2010) and is enhanced by pro-inflammatory mediators (Liuzzo et al. 1999a, b). CX3CL1 exerts its biological effects by binding to CX3CR1 for which it is the only ligand. Expression analysis of CX3CR1 has demonstrated its mRNA to be abundant in the spleen and peripheral blood leukocytes as well as the brain. Furthermore, the expression of CX3CR1 mRNA is tenfold higher in cultured microglial than whole brain samples, suggesting the receptor is of microglial origin. FACS analysis and calcium imaging of microglia have demonstrated that these cells possess the functional CX3CR1 protein as well as the mRNA (Harrison et al. 1998). This expression profile of CX3CR1 has been confirmed conclusively by the development of a transgenic mouse in which the CX3CR1 contains a green fluorescent protein reporter allowing visualisation of the transcribed protein; here, in the mouse, CX3CR1 protein was observed in microglia throughout the CNS but was not present in astrocytes or oligodendrocytes (Jung et al. 2000).

Within the CNS the CX3CL1–CX3CR1 interaction contributes the maintenance of a quiescent phenotype in microglia and suppresses the release of pro-inflammatory mediators (Mizuno et al. 2003; Lyons et al. 2009). As such, under these conditions, this protein–protein relationship is thought to be neuroprotective. In the context of chronic pain, several observations support a pro-nociceptive role of CX3CL1. The administration of the soluble chemokine domain of CX3CL1 into the intrathecal space at the lumbar level is pro-nociceptive and causes otherwise naive animals to exhibit nocifensive behaviours (Zhuang et al. 2007; Clark et al. 2007b; Milligan et al. 2004, 2005a). Consistently, the intrathecal administration of anti-CX3CL1 antibodies to neuropathic rodents attenuates pain-related behaviour (Clark et al. 2007b). Interestingly, the CSF levels of CX3CL1 increase in neuropathic animals compared to sham controls (Clark et al. 2007b, 2009). CX3CR1 exhibits similar pro-nociceptive attributes under aberrant pain conditions. Enhanced expression of the protein within the dorsal horn of the spinal cord is associated with microgliosis following peripheral nerve injury (Zhuang et al. 2007; Staniland et al. 2010). One mechanism described is IL-6 dependent; IL-6 mRNA and protein expression is induced in neurons following the peripheral nerve injury (Arruda et al. 1998; Lee et al. 2009), and prophylactic treatment with an IL-6 neutralising antibody prevents increased CX3CR1 expression, whilst, conversely, the administration of recombinant IL-6 significantly augments CX3CR1 expression (Lee et al. 2010). Supporting a pro-nociceptive role of CX3CR1 and a critical role for CX3CL1–CX3CR1 interaction in the development of pathological pain responses, CX3CR1-deficient mice do not develop hyperalgesia and/or allodynia in models of nerve injury and exhibit reduced microgliosis when compared to their wild-type littermate controls (Staniland et al. 2010). Similarly, the intrathecal administration of an anti-CX3CR1 antibody attenuates both the behavioural and microglial responses to injury (Zhuang et al. 2007; Milligan et al. 2004, 2005a). Within spinal cord microglia activation of CX3CR1 by CX3CL1 results in increased intracellular calcium concentrations (Harrison et al. 1998), the phosphorylation of p38 MAPK and subsequent release of pro-nociceptive molecules such as IL-6, NO and IL-1β (Zhuang et al. 2007; Clark et al. 2007b).

TNF, previously known as TNFα, is a small pro-inflammatory cytokine first described in activated macrophages as a molecule with tumour-regression activity (Carswell et al. 1975). TNF belongs to a superfamily of ligand/receptor proteins that share a structural motif — the TNF homology domain. The TNF receptors are the other members of this family of proteins; two have been identified and are either constitutively expressed (TNFR1/p55-R) or inducible (TNFR2/p75-R) (Bodmer et al. 2002; Leung and Cahill 2010). Under physiological conditions TNF is expressed at very low levels in the spinal cord; however, it is rapidly upregulated in both microglia and astrocytes (glia) and neurons following peripheral injury (DeLeo et al. 1997; Ohtori et al. 2004; Hao et al. 2007; Youn et al. 2008). Similarly both TNFR1 and TNFR2 are expressed within glia and neurons in the spinal cord (Gruber-Schoffnegger et al. 2013; Ohtori et al. 2004; Hao et al. 2007). The use of receptor-specific protein ligands has demonstrated that it is TNFR1 activation in the spinal cord that is responsible for the pro-nociceptive effects of TNF under steady-state conditions; TNFR2, on the other hand, contributes to the pro-nociceptive effects of TNF under chronic pain states following its upregulation after nerve injury (Liu et al. 2007; Clark et al. 2013).

In the context of peripheral nerve injury models of neuropathic pain, TNF is detectable in the periphery at the site of injury where it is upregulated and exerts pro-nociceptive functions (George et al. 1999; Shubayev and Myers 2000; Sommer and Schafers 1998; Leung and Cahill 2010). Within the spinal cord TNF exerts its pro-nociceptive effects via actions on both neurons and glia. The spinal administration of TNF is associated with the development of mechanical allodynia and thermal hypersensitivity (Youn et al. 2008; Zhang et al. 2011). Additionally the application of exogenous TNF to spinal cord slices induces rapid modulation of Aδ- and C-fibre (nociceptive-fibre)-mediated neurotransmission (Youn et al. 2008), enhances dorsal horn neuronal responses to C-fibre stimulation (Reeve et al. 2000) and augments excitatory neurotransmission in lamina II neurons in a predominantly TNFR1-dependent manner (Zhang et al. 2011). Additionally long-term potentiation (LTP) induced by stimulation of the sciatic nerve is attenuated in TNFR knock-out mice (Park et al. 2011). These effects may not be mediated directly by the activation of neuronal TNF receptors, but rather indirectly by the activation of receptors located on glial cells within the dorsal horn (Gruber-Schoffnegger et al. 2013). Indeed, TNF is also able to modulate glial cell activation within the spinal cord as blockade of TNF signalling is associated with a reduction in glial cell activity in models of peripheral nerve injury (Svensson et al. 2005b; Nadeau et al. 2011). Via P2X7 signalling ATP is able to induce TNF production in microglia by a P38 MAPK-dependent pathway (Suzuki et al. 2004; Lister et al. 2007); in rats with a peripheral nerve injury, concomitant increases of TNF and p-P38 are observed in the spinal cord, and blockade of TNF suppresses P38 phosphorylation and microgliosis (Schafers et al. 2003; Marchand et al. 2009). Evidence from cultured astrocytes demonstrates that TNF transiently activates JNK (Gao et al. 2009), a mitogen-activated kinase (MAPK) associated with the synthesis and release of pro-nociceptive mediators (Ji et al. 2009). In particular this pathway is associated with the upregulation of astrocytic CCL2; CCL2 upregulation by TNF is dose dependently inhibited by a JNK inhibitor (Gao et al. 2009). Similarly, the spinal injection of TNF produces JNK-dependent behavioural hypersensitivity, whilst the administration of a JNK inhibitor to nerve-injured animals suppresses pain-associated behaviour (Gao et al. 2009). Peripheral nerve injury induces a slow but persistent activation of JNK MAPK particularly within astrocytes in the spinal cord. Whilst the administration of a specific JNK inhibitor has no effect on acute pain responses, it potently prevents and reverses nerve injury-induced mechanical allodynia in rodents (Zhuang et al. 2006).

Interleukin-1β (IL-1β) is a small pro-inflammatory cytokine first described as a pyrogenic factor released from leukocytes (Dinarello 2007), and was one of the first cytokines to be implicated in the mechanisms underlying enhanced nociception in rodents following peripheral nerve injury (Clark et al. 2013). IL-1β, a 17.5 kDa protein, is part of the well-characterised IL-1 family of proteins that also includes IL-1α, the IL-1 receptor (IL-1R1), a decoy receptor (IL-1R2) and the endogenous receptor antagonist (IL-1ra). IL-1ra is structurally similar to IL-1β and binds to IL-1R1 with similar affinity; however, it lacks the ability to activate the receptor and stimulate downstream signalling. Similarly, IL-1R2 is able to bind IL-1β with comparable affinity to IL-1R1 but lacks the appropriate cytosolic region to activate downstream signalling proteins (Weber et al. 2010; Dunn et al. 2001). Within the CNS IL-1β is expressed at low levels by both neurons and glial cells (Ren and Torres 2009; Clark et al. 2006; Copray et al. 2001; Guo et al. 2007; Sommer and Kress 2004), and the expression of IL-1R1 is equally widespread within the spinal cord (Zhang et al. 2008; Sweitzer et al. 1999; Gruber-Schoffnegger et al. 2013).

Under physiological conditions IL-1β has a number of homeostatic functions such as regulation of sleep and temperature (Dinarello 1996). More importantly, IL-1β is a key regulator of pro-inflammatory responses and the control of innate immune responses to pathogen-associated danger signals [pathogen-associated molecular patterns (PAMPS) and danger-associated molecular patterns (DAMPS)], such as lipopolysaccharide (LPS) and bacterial DNA. The mechanism by which IL-1β is secreted from cells differs from the classical ER–Golgi route of protein secretion due to a lack of a leader sequence (Rubartelli et al. 1990). Rather, IL-1β is synthesised as a larger precursor protein and is cleaved into a mature form; the mechanism behind this has now been elucidated and involves caspase-1, which cleaves IL-1β, and a series of accessory proteins that form a complex known as the inflammasome that provides a platform for the activation of caspase (Martinon et al. 2002; Schroder and Tschopp 2010).

Similarly to TNF, IL-1β is pro-nociceptive when administered centrally; the intrathecal administration of exogenous IL-1β results in thermal and mechanical hypersensitivity (Gruber-Schoffnegger et al. 2013; Reeve et al. 2000; Sung et al. 2004; Kawasaki et al. 2008). Further evidence for a pro-nociceptive effect of IL-1β comes from rodents lacking this protein which exhibit attenuated behavioural responses to peripheral nerve injury (Wolf et al. 2006). Furthermore it has been observed that human patients with a range of painful neuropathies exhibit enhanced CSF levels of IL-1β (Alexander et al. 2005; Backonja et al. 2008). Following peripheral nerve injury, IL-1β is upregulated in the spinal cord where it is expressed primarily by glial cells but also by neurons (Clark et al. 2013). Inhibition of normal IL-1β signalling is anti-nociceptive in animal models of neuropathic pain; intrathecal administration of IL-1ra can both prevent and reverse (Milligan et al. 2005b, 2006) nocifensive behaviours in these animals, as can inhibition of caspase-1 which prevents the proteolytic activation of IL-1β, resulting in decreased secretion of the mature form from microglia (Clark et al. 2006).

IL-1β released from glial cells is able to modulate neuronal activity. This cytokine can facilitate glutamatergic transmission via the phosphorylation of the NMDA receptor (Gruber-Schoffnegger et al. 2013) which enhances behavioural hypersensitivity; treatment with IL-1ra attenuates both the behavioural phenotype and receptor phosphorylation. The mechanism underlying this modulation is complex and calcium dependent (Viviani et al. 2003), and involves several intracellular signalling proteins including PKC, IP3, PLC, PLA2 and src kinases. Inhibitors of these proteins are able to block IL-1β-induced phosphorylation of either the NR1 subunit or NR2A/B subunit of the NMDA receptor in vitro similarly to IL-1ra (Viviani et al. 2003). The application of exogenous IL-1β to spinal cord slices is able to increase AMPA-mediated currents in lamina II neurons (Kawasaki et al. 2008) and induce LTP within lamina I neurons (Gruber-Schoffnegger et al. 2013). Patch clamp studies of IL-1β-treated spinal cord slices demonstrate that IL-1β enhances excitatory neurotransmission whilst attenuating inhibitory neurotransmission (Kawasaki et al. 2008). Overall these data indicate that IL-1β contributes to a potentiation of excitatory transmission and a suppression of inhibitory transmission, which together correlate with the facilitation of behavioural hypersensitivity.

As with many other cytokines, as well as a neuronal effect, IL-1β is thought to contribute to a pro-nociceptive state via the activation of glia. Additionally glia may contribute to the effects of IL-1β on neuronal excitability as these are absent following the inhibition of glial cell function (Gruber-Schoffnegger et al. 2013; Liu et al. 2013).