Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.     

Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats

Recent evidence suggests that spinal cord glia can contribute to enhanced nociceptive responses. However, the signals that cause glial activation are unknown. Fractalkine (CX3C ligand-1; CX3CL1) is a unique chemokine expressed on the extracellular surface of spinal neurons and spinal sensory afferents. In the dorsal spinal cord, fractalkine receptors are primarily expressed by microglia. As fractalkine can be released from neurons upon strong activation, it has previously been suggested to be a neuron-to-glial signal that induces glial activation. The present series of experiments provide an initial investigation of the spinal pain modulatory effects of fractalkine. Intrathecal fractalkine produced dose-dependent mechanical allodynia and thermal hyperalgesia. In addition, a single injection of fractalkine receptor antagonist (neutralizing antibody against rat CX3C receptor-1; CX3CR1) delayed the development of mechanical allodynia and/or thermal hyperalgesia in two neuropathic pain models: chronic constriction injury (CCI) and sciatic inflammatory neuropathy. Intriguingly, anti-CX3CR1 reduced nociceptive responses when administered 5-7 days after CCI, suggesting that prolonged release of fractalkine may contribute to the maintenance of neuropathic pain. Taken together, these initial investigations of spinal fractalkine effects suggest that exogenous and endogenous fractalkine are involved in spinal sensitization, including that induced by peripheral neuropathy.     

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions

Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron-to-glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up-regulated in a regionally specific manner in response to neuropathy. CX3CR1-expressing cells were identified as microglia by their cellular morphology and positive OX-42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.     

Glial activation in the periaqueductal gray promotes descending facilitation of neuropathic pain through the p38 MAPK signaling pathway

The midbrain ventrolateral periaqueductal gray (VL‐PAG) is a key component that mediates pain modulation. Although spinal cord glial cells appear to play an important role in chronic pain development, the precise mechanisms involving descending facilitation pathways from the PAG following nerve injury are poorly understood. This study shows that cellular events that occur during glial activation in the VL‐PAG may promote descending facilitation from the PAG during neuropathic pain. Chronic constriction nerve injury (CCI) was induced by ligature construction of the sciatic nerve in male Sprague‐Dawley rats. Behavioral responses to noxious mechanical (paw withdrawal threshold; PWT) and thermal (paw withdrawal latency; PWL) stimuli were evaluated. After CCI, immunohistochemical and Western blot analysis of microglia and astrocytes in the VL‐PAG showed morphological and quantitative changes indicative of activation in microglia and astrocytes. Intra‐VL‐PAG injection of microglial or astrocytic inhibitors attenuated PWT and PWL at days 7 and 14, respectively, following CCI. We also evaluated the effects of intra‐VL‐PAG administration of the phosphorylated p38 mitogen‐activated protein kinase (p‐p38 MAPK) inhibitor SB 203580 at day 7 after CCI. This treatment abolished microglial activation and produced a significant time‐dependent attenuation of PWT and PWL. Western blot analysis showed localized expression of p‐p38 in the VL‐PAG after CCI. P‐p38 was expressed in labeled microglia of the VL‐PAG but was not present in astrocytes and neurons on day 7 after CCI. These results demonstrate that CCI‐induced neuropathic pain is associated with glial activation in the VL‐PAG, which likely participates in descending pain facilitation through the p38 MAPK signaling pathway. © 2015 Wiley Periodicals, Inc.     

Sex-Dependent Glial Signaling in Pathological Pain: Distinct Roles of Spinal Microglia and Astrocytes

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID(a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate(L-AA, an astroglial toxin) and carbenoxolone(a connexin 43 inhibitor), as well as U0126(an ERK kinase inhibitor) and D-JNKI-1(a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast,intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.     

CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway

Peripheral nerve injury induces the cleavage of CX3CL1 from the membrane of neurons, where the soluble CX3CL1 subsequently plays an important role in the transmission of nociceptive signals between neurons and microglia. Here we investigated whether CX3CL1 regulates microglia activation through the phosphorylation of extracellular signal‐regulated protein kinase 5 (ERK5) in the spinal cord of rats with spinal nerve ligation (SNL). ERK5 and microglia were activated in the spinal cord after SNL. The knockdown of ERK5 by intrathecal injection of antisense oligonucleotides suppressed the hyperalgesia and nuclear impact of nuclear factor‐κB induced by SNL. The blockage of CX3CR1, the receptor of CX3CL1, significantly reduced the level of ERK5 activation following SNL. In addition, the antisense knockdown of ERK5 reversed the CX3CL1‐induced hyperalgesia and spinal microglia activation. Our study suggests that CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway. © 2013 Wiley Periodicals, Inc.     

Activation of cannabinoid receptor 2 attenuates mechanical allodynia and neuroinflammatory responses in a chronic post‐ischemic pain model of complex regional pain syndrome type I in rats

Complex regional pain syndrome type 1 (CRPS‐I) remains one of the most clinically challenging neuropathic pain syndromes and its mechanism has not been fully characterized. Cannabinoid receptor 2 (CB2) has emerged as a promising target for treating different neuropathic pain syndromes. In neuropathic pain models, activated microglia expressing CB2 receptors are seen in the spinal cord. Chemokine fractalkine receptor (CX3CR1) plays a substantial role in microglial activation and neuroinflammation. We hypothesized that a CB2 agonist could modulate neuroinflammation and neuropathic pain in an ischemia model of CRPS by regulating CB2 and CX3CR1 signaling. We used chronic post‐ischemia pain (CPIP) as a model of CRPS‐I. Rats in the CPIP group exhibited significant hyperemia and edema of the ischemic hindpaw and spontaneous pain behaviors (hindpaw shaking and licking). Intraperitoneal administration of MDA7 (a selective CB2 agonist) attenuated mechanical allodynia induced by CPIP. MDA7 treatment was found to interfere with early events in the CRPS‐I neuroinflammatory response by suppressing peripheral edema, spinal microglial activation and expression of CX3CR1 and CB2 receptors on the microglia in the spinal cord. MDA7 also mitigated the loss of intraepidermal nerve fibers induced by CPIP. Neuroprotective effects of MDA7 were blocked by a CB2 antagonist, AM630. Our findings suggest that MDA7, a novel CB2 agonist, may offer an innovative therapeutic approach for treating neuropathic symptoms and neuroinflammatory responses induced by CRPS‐I in the setting of ischemia and reperfusion injury.     

RhoA/ROCK pathway mediates p38 MAPK activation and morphological changes downstream of P2Y12/13 receptors in spinal microglia in neuropathic pain

Recent studies have indicated an important role of ATP receptors in spinal microglia, such as P2Y12 or P2Y13, in the development of chronic pain. However, intracellular signaling cascade of these receptors have not been clearly elucidated. We found that intrathecal injection of 2‐(methylthio)adenosine 5′‐diphosphate (2Me‐SADP) induced mechanical hypersensitivity and p38 mitogen‐activated protein kinase (MAPK) phosphorylation in the spinal cord. Intrathecal administration of P2Y12/P2Y13 antagonists and Rho‐associated coiled‐coil‐containing protein kinase (ROCK) inhibitor H1152 suppressed not only p38 MAPK phosphorylation, but also mechanical hypersensitivity induced by 2Me‐SADP. In the rat peripheral nerve injury model, intrathecal administration of antagonists for the P2Y12/P2Y13 receptor suppressed activation of p38 MAPK in the spinal cord. In addition, subarachnoidal injection of H1152 also attenuated nerve injury‐induced spinal p38 MAPK phosphorylation and neuropathic pain behavior, suggesting an essential role of ROCK in nerve injury‐induced p38 MAPK activation. We also found that the antagonists of the P2Y12/P2Y13 receptor and H1152 had inhibitory effects on the morphological changes of microglia such as retraction of processes in both 2Me‐SADP and nerve injured rats. In contrast these treatments had no effect on the number of Iba1‐positive cells in the nerve injury model. Collectively, our results have demonstrated roles of ROCK in the spinal microglia that is involved in p38 MAPK activation and the morphological changes. Inhibition of ROCK signaling may offer a novel target for the development of a neuropathic pain treatment. GLIA 2015;63:216–228     

COX‐1‐dependent prostaglandin D2 in microglia contributes to neuropathic pain via DP2 receptor in spinal neurons

Cyclooxygenase (COX) enzyme synthesizes prostaglandins (PGs) from arachidonic acid and exists as two major isozymes, COX‐1 and COX‐2. The crucial role of prostaglandins in the pathogenesis of inflammatory pain in peripheral tissue and the spinal cord has been established; however its expression dynamics after peripheral nerve injury and its role in neuropathic pain are not clear. In this study, we examined the detailed expression patterns of genes for COX, PGD2 and thromboxane A2 synthases and their receptors in the spinal cord. Furthermore, we explored the altered gene expression of these molecules using the spared nerve injury (SNI) model. We also examined whether these molecules have a role in the development or maintenance of neuropathic pain. We found a number of interesting results in this study, the first was that COX‐1 was constitutively expressed in the spinal cord and up‐regulated in microglia located in laminae I‐II after nerve injury. Second, COX‐2 mRNA expression was induced in blood vessels after nerve injury. Third, TXA2 synthase and hematopoietic PGD synthase mRNAs were dramatically increased in the microglia after nerve injury. Finally, we found that intrathecal injection of a COX‐1 inhibitor and DP2 receptor antagonist significantly attenuated the mechanical allodynia. Our findings indicate that PGD2 produced by microglia is COX‐1 dependent, and that neurons in the spinal cord can receive PGD2 from microglia following peripheral nerve injury. We believe that PGD2 signaling via DP2 signaling pathway from microglia to neurons is one of the triggering factors for mechanical allodynia in this neuropathic pain model.     

Involvement of NF-κB and the CX3CR1 Signaling Network in Mechanical Allodynia Induced by Tetanic Sciatic Stimulation

Tetanic stimulation of the sciatic nerve(TSS)triggers long-term potentiation in the dorsal horn of the spinal cord and long-lasting pain hypersensitivity. CX3CL1-CX3CR1 signaling is an important pathway in neuronalmicroglial activation. Nuclear factor κB(NF-κB) is a key signal transduction molecule that regulates neuroinflammation and neuropathic pain. Here, we set out to determine whether and how NF-κB and CX3CR1 are involved in the mechanism underlying the pathological changes induced by TSS. After unilateral TSS, significant bilateral mechanical allodynia was induced, as assessed by the von Frey test. The expression of phosphorylated NF-κB(pNF-κB) and CX3CR1 was significantly up-regulated in the bilateral dorsal horn. Immunofluorescence staining demonstrated that pNF-KB and NeuN co-existed, implying that the NF-κB pathway is predominantly activated in neurons following TSS. Administration of either the NF-κB inhibitor ammonium pyrrolidine dithiocarbamate or a CX3CR1-neutralizing antibody blocked the development and maintenance of neuropathic pain. In addition, blockade of NF-κB downregulated the expression of CX3 CL1-CX3CR1 signaling,and conversely the CX3CR1-neutralizing antibody also down-regulated pNF-κB. These findings suggest an involvement of NF-κB and the CX3CR1 signaling network in the development and maintenance of TSS-induced mechanical allodynia. Our work suggests the potential clinical application of NF-κB inhibitors or CX3CR1-neutralizing antibodies in treating pathological pain.     

Spinal P2X(7) receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model

P2X₇ receptor is an important member of ATP-sensitive ionotropic P2X receptors family, which includes seven receptor subtypes (P2X₁-P2X₇). Recent evidence indicates that P2X₇R participates in the onset and persistence of neuropathic pain. In tetanic stimulation of the sciatic nerve model, P2X₇R was involved in the activation of microglia, but whether this happens in other neuropathic pain models remains unclear. In this study we used immunohistochemistry and Western blot to explore the relationship of P2X₇R expression with microglia activation, and with mechanical allodynia and thermal hypersensitivity in the chronic constriction of the sciatic nerve (CCI) rat model. The results show that following nerve ligature, mechanical allodynia and thermal hypersensitivity were developed within 3 days (d), peaked at 14 d and persisted for 21 d on the injured side. P2X₇R levels in the ipsilateral L4-6 spinal cord were increased markedly after injury and the highest levels were observed on day 14, significant difference was observed at I-IV layers of the dorsal horn. The change in P2X₇R levels in the spinal cord was consistent with the development of mechanical allodynia and thermal hypersensitivity. Intrathecal administration of the P2X₇R antagonist Brilliant Blue G (BBG) reversed CCI-induced mechanical allodynia and thermal hypersensitivity. Double-labeled immunofluorescence showed that P2X₇R expression were restricted to microglia, spinal microglia were activated after nerve injury, which was inhibited by BBG. These results indicated that spinal P2X₇R mediate microglia activation, this process may play an important role in development of mechanical allodynia and thermal hypersensitivity in CCI model.     

Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury

Recent work regarding chronic central neuropathic pain (CNP) following spinal cord injury (SCI) suggests that activation of key signaling molecules such as members of the mitogen activated protein kinase (MAPK) family play a role in the expression of at-level mechanical allodynia. Previously, we have shown that the development of at-level CNP following moderate spinal cord injury is correlated with increased expression of the activated (and thus phosphorylated) forms of the MAPKs extracellular signal related kinase and p38 MAPK. The current study extends this work by directly examining the role of p38 MAPK in the maintenance of at-level CNP following spinal cord injury. Using a combination of behavioral, immunocytochemical, and electrophysiological measures we demonstrate that increased activation of p38 MAPK occurs in the spinal cord just rostral to the site of injury in rats that develop at-level mechanical allodynia after moderate SCI. Immunocytochemical analyses indicate that the increases in p38 MAPK activation occurred in astrocytes, microglia, and dorsal horn neurons in the spinal cord rostral to the site of injury. Inhibiting the enzymatic activity of p38 MAPK dose dependently reverses the behavioral expression of at-level mechanical allodynia and also decreases the hyperexcitability seen in thoracic dorsal horn neurons after moderate SCI. Taken together, these novel data are the first to demonstrate causality that increased activation of p38 MAPK in multiple cell types play an important role in the maintenance of at-level CNP following spinal cord injury.     

Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling

A single intrathecal dose of adenosine 2A receptor (A_2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A_2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A₁R and A_2BR agonist in CCI and found the same long duration effect with A_2BR but not A₁R agonism. An A_2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A_2AR agonist on neuropathic allodynia. Therefore, A_2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.     

miR-26a-5p alleviates CFA-induced chronic inflammatory hyperalgesia through Wnt5a/CaMKII/NFAT signaling in mice

Background

Inflammation often leads to the occurrence of chronic pain, and many miRNAs have been shown to play a key role in the development of inflammatory pain. However, whether miR-26a-5p relieves pain induced by inflammation and its possible mechanism are still unclear.

Methods

The complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model was employed. Intrathecal or subcutaneous injection of miR-26a-5p agomir was performed after modeling to study its antinociceptive effect and the comparison of different administration methods. Bioinformatics analysis of miRNAs was performed to study the downstream mechanisms of miR-26a-5p. HE staining, RT-qPCR, Western blotting, and immunofluorescence were used for further validation.

Results

A single intrathecal and subcutaneous injection of miR-26a-5p both reversed mechanical hypersensitivity and thermal latency in the left hind paw of mice with CFA-induced inflammatory pain. HE staining and immunofluorescence studies found that both administrations of miR-26a-5p alleviated inflammation in the periphery and spinal cord. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. Wnt5a was mainly expressed in neurons and microglia in the spinal cord of mice with inflammatory pain. Intrathecal injection of miR-26a-5p could significantly reduce the expression level of Wnt5a and inhibit the downstream molecules of noncanonical Wnt signaling Camk2/NFAT, inhibiting the release of spinal cord inflammatory factors and alleviating the activation of microglia. In addition, miR-26a-5p could also inhibit lipopolysaccharide (LPS)-stimulated BV2 cell inflammation in vitro through a noncanonical Wnt signaling pathway.

Conclusions

miR-26a-5p is a promising therapy for CFA-induced inflammatory pain. Both intrathecal and subcutaneous injections provide relief for inflammatory pain. miR-26a-5p regulated noncanonical Wnt signaling to be involved in analgesia partly through antineuroinflammation, suggesting a pain-alleviating effect via noncanonical Wnt signaling pathway in the CFA-induced inflammatory pain model in vivo.     

Spinal cord Toll-like receptor 4 mediates inflammatory and neuropathic hypersensitivity in male but not female mice

The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here, using pharmacological and genetic manipulations, we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freund's adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4 dependent in males but TLR4 independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 gene expression at baseline or after LPS, suggesting the existence of parallel spinal pain-processing circuitry in female mice not involving TLR4.     

CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.     

Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration

Peripheral nerve injury activates spinal glial cells, which may contribute to the development of pain behavioral hypersensitivity. There is growing evidence that activated microglia show dynamic changes in cell morphology; however, the molecular mechanisms that underlie the modification of the membrane and cytoskeleton of microglia are not known. Here, we investigated the phosphorylation of ezrin, radixin, and moesin (ERM) proteins in the spinal cord after peripheral nerve injury. ERM is known to function as membrane‐cytoskeletal linkers and be localized at filopodia‐ and microvilli‐like structures. ERM proteins must be phosphorylated at a specific C‐terminal threonine residue to be in the active state. The nature of ERM proteins in the spinal cord of animals in a neuropathic pain model has not been investigated and characterized. In the present study, we observed an increase in the phosphorylated ERM in the spinal microglia following spared nerve injury. The intrathecal administration of lysophosphatidic acid induced the phosphorylation of ERM proteins in microglia along with the development of mechanical pain hypersensitivity. Intrathecal administration of ERM antisense locked nucleic acid suppressed nerve injury‐induced tactile allodynia and decreased the phosphorylation of ERM, but not the Iba1 staining pattern, in spinal glial cells. These findings suggest that lysophosphatidic acid induced the phosphorylation of ERM proteins in spinal microglia and may be involved in the emergence of neuropathic pain. These findings may underlie the pathological mechanisms of nerve injury‐induced neuropathic pain. © 2012 Wiley Periodicals, Inc.     

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine-induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin-1 (IL1), interleukin-6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine-induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti-CX3CR1 also blocked fractalkine-induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine-induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine-induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine-induced pain facilitation. Lastly, NO appears to be importantly involved, as l-NAME, a broad-spectrum NO synthase inhibitor, also blocked fractalkine-induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron-to-glia signal triggering pain facilitation.