Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

Differential involvement of trigeminal transition zone and laminated subnucleus caudalis in orofacial deep and cutaneous hyperalgesia: the effects of interleukin-10 and glial inhibitors

Background

Understanding the underlying mechanisms of neuropathic pain caused by damage to the peripheral nervous system remains challenging and could lead to significantly improved therapies. Disturbance of homeostasis not only occurs at the site of injury but also extends to the spinal cord and brain involving various types of cells. Emerging data implicate neuroimmune interaction in the initiation and maintenance of chronic pain hypersensitivity.

Results

In this study, we sought to investigate the effects of TGF-β1, a potent anti-inflammatory cytokine, in alleviating nerve injury-induced neuropathic pain in rats. By using a well established neuropathic pain animal model (partial ligation of the sciatic nerve), we demonstrated that intrathecal infusion of recombinant TGF-β1 significantly attenuated nerve injury-induced neuropathic pain. TGF-β1 treatment not only prevents development of neuropathic pain following nerve injury, but also reverses previously established neuropathic pain conditions. The biological outcomes of TGF-β1 in this context are attributed to its pleiotropic effects. It inhibits peripheral nerve injury-induced spinal microgliosis, spinal microglial and astrocytic activation, and exhibits a powerful neuroprotective effect by preventing the induction of ATF3⁺ neurons following nerve ligation, consequently reducing the expression of chemokine MCP-1 in damaged neurons. TGF-β1 treatment also suppresses nerve injury-induced inflammatory response in the spinal cord, as revealed by a reduction in cytokine expression.

Conclusion

Our findings revealed that TGF-β1 is effective in the treatment of neuropathic by targeting both neurons and glial cells. We suggest that therapeutic agents such as TGF-β1 having multipotent effects on different types of cells could work in synergy to regain homeostasis in local spinal cord microenvironments, therefore contributing to attenuate neuropathic pain.     

Roles of extracellular signal-regulated protein kinases 5 in spinal microglia and primary sensory neurons for neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.     

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

Tramadol and Propentofylline Coadministration Exerted Synergistic Effects on Rat Spinal Nerve Ligation-Induced Neuropathic Pain

Neuropathic pain is an intractable clinical problem. Drug treatments such as tramadol have been reported to effectively decrease neuropathic pain by inhibiting the activity of nociceptive neurons. It has also been reported that modulating glial activation could also prevent or reverse neuropathic pain via the administration of a glial modulator or inhibitor, such as propentofylline. Thus far, there has been no clinical strategy incorporating both neuronal and glial participation for treating neuropathic pain. Therefore, the present research study was designed to assess whether coadministration of tramadol and propentofylline, as neuronal and glial activation inhibitors, respectively, would exert a synergistic effect on the reduction of rat spinal nerve ligation (SNL)-induced neuropathic pain. Rats underwent SNL surgery to induce neuropathic pain. Pain behavioral tests were conducted to ascertain the effect of drugs on SNL-induced mechanical allodynia with von-Frey hairs. Proinflammatory factor interleukin-1β (IL-1β) expression was also detected by Real-time RT-PCR. Intrathecal tramadol and propentofylline administered alone relieved SNL-induced mechanical allodynia in a dose-dependent manner. Tramadol and propentofylline coadministration exerted a more potent effect in a synergistic and dose dependent manner than the intrathecal administration of either drug alone. Real-time RT-PCR demonstrated IL-1β up-expression in the ipsilateral spinal dorsal horn after the lesion, which was significantly decreased by tramadol and propentofylline coadministration. Inhibiting proinflammatory factor IL-1β contributed to the synergistic effects of tramadol and propentofylline coadministration on rat peripheral nerve injury-induced neuropathic pain. Thus, our study provided a rationale for utilizing a novel strategy for treating neuropathic pain by blocking the proinflammatory factor related pathways in the central nervous system.     

Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain

Treatment of neuropathic pain, triggered by multiple insults to the nervous system, is a clinical challenge because the underlying mechanisms of neuropathic pain development remain poorly understood. Most treatments do not differentiate between different phases of neuropathic pain pathophysiology and simply focus on blocking neurotransmission, producing transient pain relief. Here, we report that early- and late-phase neuropathic pain development in rats and mice after nerve injury require different matrix metalloproteinases (MMPs). After spinal nerve ligation, MMP-9 shows a rapid and transient upregulation in injured dorsal root ganglion (DRG) primary sensory neurons consistent with an early phase of neuropathic pain, whereas MMP-2 shows a delayed response in DRG satellite cells and spinal astrocytes consistent with a late phase of neuropathic pain. Local inhibition of MMP-9 by an intrathecal route inhibits the early phase of neuropathic pain, whereas inhibition of MMP-2 suppresses the late phase of neuropathic pain. Further, intrathecal administration of MMP-9 or MMP-2 is sufficient to produce neuropathic pain symptoms. After nerve injury, MMP-9 induces neuropathic pain through interleukin-1beta cleavage and microglial activation at early times, whereas MMP-2 maintains neuropathic pain through interleukin-1beta cleavage and astrocyte activation at later times. Inhibition of MMP may provide a novel therapeutic approach for the treatment of neuropathic pain at different phases.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Role of Lipocalin-2-Chemokine Axis in the Development of Neuropathic Pain following Peripheral Nerve Injury

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.     

Peripheral Nerve Injury Modulates Neurotrophin Signaling in the Peripheral and Central Nervous System

Peripheral nerve injury disrupts the normal functions of sensory and motor neurons by damaging the integrity of axons and Schwann cells. In contrast to the central nervous system, the peripheral nervous system possesses a considerable capacity for regrowth, but regeneration is far from complete and functional recovery rarely returns to pre-injury levels. During development, the peripheral nervous system strongly depends upon trophic stimulation for neuronal differentiation, growth and maturation. The perhaps most important group of trophic substances in this context is the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), which signal in a complex spatial and timely manner via the two structurally unrelated p75^NTR and tropomyosin receptor kinase (TrkA, Trk-B and Trk-C) receptors. Damage to the adult peripheral nerves induces cellular mechanisms resembling those active during development, resulting in a rapid and robust increase in the synthesis of neurotrophins in neurons and Schwann cells, guiding and supporting regeneration. Furthermore, the injury induces neurotrophin-mediated changes in the dorsal root ganglia and in the spinal cord, which affect the modulation of afferent sensory signaling and eventually may contribute to the development of neuropathic pain. The focus of this review is on the expression patterns of neurotrophins and their receptors in neurons and glial cells of the peripheral nervous system and the spinal cord. Furthermore, injury-induced changes of expression patterns and the functional consequences in relation to axonal growth and remyelination as well as to neuropathic pain development will be reviewed.     

Persistent At-Level Thermal Hyperalgesia and Tactile Allodynia Accompany Chronic Neuronal and Astrocyte Activation in Superficial Dorsal Horn following Mouse Cervical Contusion Spinal Cord Injury

In humans, sensory abnormalities, including neuropathic pain, often result from traumatic spinal cord injury (SCI). SCI can induce cellular changes in the CNS, termed central sensitization, that alter excitability of spinal cord neurons, including those in the dorsal horn involved in pain transmission. Persistently elevated levels of neuronal activity, glial activation, and glutamatergic transmission are thought to contribute to the hyperexcitability of these dorsal horn neurons, which can lead to maladaptive circuitry, aberrant pain processing and, ultimately, chronic neuropathic pain. Here we present a mouse model of SCI-induced neuropathic pain that exhibits a persistent pain phenotype accompanied by chronic neuronal hyperexcitability and glial activation in the spinal cord dorsal horn. We generated a unilateral cervical contusion injury at the C5 or C6 level of the adult mouse spinal cord. Following injury, an increase in the number of neurons expressing ΔFosB (a marker of chronic neuronal activation), persistent astrocyte activation and proliferation (as measured by GFAP and Ki67 expression), and a decrease in the expression of the astrocyte glutamate transporter GLT1 are observed in the ipsilateral superficial dorsal horn of cervical spinal cord. These changes have previously been associated with neuronal hyperexcitability and may contribute to altered pain transmission and chronic neuropathic pain. In our model, they are accompanied by robust at-level hyperaglesia in the ipsilateral forepaw and allodynia in both forepaws that are evident within two weeks following injury and persist for at least six weeks. Furthermore, the pain phenotype occurs in the absence of alterations in forelimb grip strength, suggesting that it represents sensory and not motor abnormalities. Given the importance of transgenic mouse technology, this clinically-relevant model provides a resource that can be used to study the molecular mechanisms contributing to neuropathic pain following SCI and to identify potential therapeutic targets for the treatment of chronic pathological pain.     

Neuropathic Pain in Rats with a Partial Sciatic Nerve Ligation Is Alleviated by Intravenous Injection of Monoclonal Antibody to High Mobility Group Box-1

High mobility group box-1 (HMGB1) is associated with the pathogenesis of inflammatory diseases. A previous study reported that intravenous injection of anti-HMGB1 monoclonal antibody significantly attenuated brain edema in a rat model of stroke, possibly by attenuating glial activation. Peripheral nerve injury leads to increased activity of glia in the spinal cord dorsal horn. Thus, it is possible that the anti-HMGB1 antibody could also be efficacious in attenuating peripheral nerve injury-induced pain. Following partial sciatic nerve ligation (PSNL), rats were treated with either anti-HMGB1 or control IgG. Intravenous treatment with anti-HMGB1 monoclonal antibody (2 mg/kg) significantly ameliorated PSNL-induced hind paw tactile hypersensitivity at 7, 14 and 21 days, but not 3 days, after ligation, whereas control IgG had no effect on tactile hypersensitivity. The expression of HMGB1 protein in the spinal dorsal horn was significantly increased 7, 14 and 21 days after PSNL; the efficacy of the anti-HMGB1 antibody is likely related to the presence of HMGB1 protein. Also, the injury-induced translocation of HMGB1 from the nucleus to the cytosol occurred mainly in dorsal horn neurons and not in astrocytes and microglia, indicating a neuronal source of HMGB1. Markers of astrocyte (glial fibrillary acidic protein (GFAP)), microglia (ionized calcium binding adaptor molecule 1 (Iba1)) and spinal neuron (cFos) activity were greatly increased in the ipsilateral dorsal horn side compared to the sham-operated side 21 days after PSNL. Anti-HMGB1 monoclonal antibody treatment significantly decreased the injury-induced expression of cFos and Iba1, but not GFAP. The results demonstrate that nerve injury evokes the synthesis and release of HMGB1 from spinal neurons, facilitating the activity of both microglia and neurons, which in turn leads to symptoms of neuropathic pain. Thus, the targeting of HMGB1 could be a useful therapeutic strategy in the treatment of chronic pain.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Possible role of spinal astrocytes in maintaining chronic pain sensitization: review of current evidence with focus on bFGF/JNK pathway

Although pain is regarded traditionally as neuronally mediated, recent progress shows an important role of spinal glial cells in persistent pain sensitization. Mounting evidence has implicated spinal microglia in the development of chronic pain (e.g. neuropathic pain after peripheral nerve injury). Less is known about the role of astrocytes in pain regulation. However, astrocytes have very close contact with synapses and maintain homeostasis in the extracellular environment. In this review, we provide evidence to support a role of spinal astrocytes in maintaining chronic pain. In particular, c-Jun N-terminal kinase (JNK) is activated persistently in spinal astrocytes in a neuropathic pain condition produced by spinal nerve ligation. This activation is required for the maintenance of neuropathic pain because spinal infusion of JNK inhibitors can reverse mechanical allodynia, a major symptom of neuropathic pain. Further study reveals that JNK is activated strongly in astrocytes by basic fibroblast growth factor (bFGF), an astroglial activator. Intrathecal infusion of bFGF also produces persistent mechanical allodynia. After peripheral nerve injury, bFGF might be produced by primary sensory neurons and spinal astrocytes because nerve injury produces robust bFGF upregulation in both cell types. Therefore, the bFGF/JNK pathway is an important signalling pathway in spinal astrocytes for chronic pain sensitization. Investigation of signaling mechanisms in spinal astrocytes will identify new molecular targets for the management of chronic pain.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

CCL-1 in the spinal cord contributes to neuropathic pain induced by nerve injury

Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However, the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. We found that CCL-1 was upregulated in the spinal dorsal horn after partial sciatic nerve ligation. Therefore, we examined actions of recombinant CCL-1 on behavioural pain score, synaptic transmission, glial cell function and cytokine production in the spinal dorsal horn. Here we show that CCL-1 is one of the key mediators involved in the development of neuropathic pain. Expression of CCL-1 mRNA was mainly detected in the ipsilateral dorsal root ganglion, and the expression of specific CCL-1 receptor CCR-8 was upregulated in the superficial dorsal horn. Increased expression of CCR-8 was observed not only in neurons but also in microglia and astrocytes in the ipsilateral side. Recombinant CCL-1 injected intrathecally (i.t.) to naive mice induced allodynia, which was prevented by the supplemental addition of N-methyl-𝒟-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that application of CCL-1 transiently enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). In the long term, i.t. injection of CCL-1 induced phosphorylation of NMDA receptor subunit, NR1 and NR2B, in the spinal cord. Injection of CCL-1 also upregulated mRNA level of glial cell markers and proinflammatory cytokines (IL-1β, TNF-α and IL-6). The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and by knocking down of CCR-8. Our results indicate that CCL-1 is one of the key molecules in pathogenesis, and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.     

Forebrain GABAergic neuron precursors integrate into adult spinal cord and reduce injury-induced neuropathic pain

Neuropathic pain is a chronic debilitating disease characterized by mechanical allodynia and spontaneous pain. Because symptoms are often unresponsive to conventional methods of pain treatment, new therapeutic approaches are essential. Here, we describe a strategy that not only ameliorates symptoms of neuropathic pain but is also potentially disease modifying. We show that transplantation of immature telencephalic GABAergic interneurons from the mouse medial ganglionic eminence (MGE) into the adult mouse spinal cord completely reverses the mechanical hypersensitivity produced by peripheral nerve injury. Underlying this improvement is a remarkable integration of the MGE transplants into the host spinal cord circuitry, in which the transplanted cells make functional connections with both primary afferent and spinal cord neurons. By contrast, MGE transplants were not effective against inflammatory pain. Our findings suggest that MGE-derived GABAergic interneurons overcome the spinal cord hyperexcitability that is a hallmark of nerve injury-induced neuropathic pain.     

Mas-related G protein-coupled receptor D participates in inflammatory pain by promoting NF-κB activation through interaction with TAK1 and IKK complex

Mas-related G protein-coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical hyperalgesia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Here, we used a mouse model of chronic inflammatory pain established by intraperitoneal administration of lipopolysaccharide (LPS). The LPS injection induced an evident peripheral neuroinflammation and mechanical hyperalgesia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. Behaviorally, the LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd-knockout mice compared with wildtype littermates. Mrgprd deletion in DRGs suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of pro-inflammatory factors. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB's DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes, but not with mitogen-activated protein kinases (MAPKs) in mouse DRGs. In macrophage-like RAW 264.7 cells, MrgprD also interacted with TAK1 and IKK complex, and the treatment of MrgprD agonist elicited the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) signaling pathway. Our findings indicate that MrgprD facilitates the development of LPS-triggered persistent inflammatory hyperalgesia by promoting canonical NF-κB activation, highlighting the important roles of MrgprD in NF-κB-mediated inflammation and chronic pain.     

Ketamine depresses toll-like receptor 3 signaling in spinal microglia in a rat model of neuropathic pain

Reports suggest that microglia play a key role in spinal nerve ligation (SNL)-induced neuropathic pain, and toll-like receptor 3 (TLR3) has a substantial role in the activation of spinal microglia and the development of tactile allodynia after nerve injury. In addition, ketamine application could suppress microglial activation in vitro, and ketamine could inhibit proinflammatory gene expression possibly by suppressing TLR-mediated signal transduction. Therefore, the present study was designed to disclose whether intrathecal ketamine could suppress SNL-induced spinal microglial activation and exert some antiallodynic effects on neuropathic pain by suppressing TLR3 activation. Behavioral results showed that intrathecal ketamine attenuated SNL-induced mechanical allodynia, as well as spinal microglial activation, in a dose-dependent manner. Furthermore, Western blot analysis displayed that ketamine application downregulated SNL-induced phosphorylated-p38 (p-p38) expression, which was specifically expressed in spinal microglia but not in astrocytes or neurons. Besides, ketamine could reverse TLR3 agonist (polyinosine-polycytidylic acid)-induced mechanical allodynia and spinal microglia activation. It was concluded that intrathecal ketamine depresses TLR3-induced spinal microglial p-p38 mitogen-activated protein kinase pathway activation after SNL, probably contributing to the antiallodynic effect of ketamine on SNL-induced neuropathic pain.     

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP⁺) BM cells (TRPM2^BM+/Rec+, TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2^BM+/Rec+ mice was attenuated in TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice. The numbers of GFP⁺ BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP⁺ BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2^BM+/Rec+ mice. However, the numbers of GFP^–/Iba1⁺ resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.