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1.  Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice 
Brain  2014;137(8):2193-2209.
Increasing evidence suggests that spinal cord astrocytes maintain neuropathic pain sensitization, but precisely how they do this is unclear. Using a mouse model of neuropathic pain, Chen et al. demonstrate that the hemichannel connexin-43 is upregulated in astrocytes after nerve injury, and maintains late-phase neuropathic pain by inducing chemokine release.
Accumulating evidence suggests that spinal cord astrocytes play an important role in neuropathic pain sensitization by releasing astrocytic mediators (e.g. cytokines, chemokines and growth factors). However, it remains unclear how astrocytes control the release of astrocytic mediators and sustain late-phase neuropathic pain. Astrocytic connexin-43 (now known as GJ1) has been implicated in gap junction and hemichannel communication of cytosolic contents through the glial syncytia and to the extracellular space, respectively. Connexin-43 also plays an essential role in facilitating the development of neuropathic pain, yet the mechanism for this contribution remains unknown. In this study, we investigated whether nerve injury could upregulate connexin-43 to sustain late-phase neuropathic pain by releasing chemokine from spinal astrocytes. Chronic constriction injury elicited a persistent upregulation of connexin-43 in spinal astrocytes for >3 weeks. Spinal (intrathecal) injection of carbenoxolone (a non-selective hemichannel blocker) and selective connexin-43 blockers (connexin-43 mimetic peptides 43Gap26 and 37,43Gap27), as well as astroglial toxin but not microglial inhibitors, given 3 weeks after nerve injury, effectively reduced mechanical allodynia, a cardinal feature of late-phase neuropathic pain. In cultured astrocytes, TNF-α elicited marked release of the chemokine CXCL1, and the release was blocked by carbenoxolone, Gap26/Gap27, and connexin-43 small interfering RNA. TNF-α also increased connexin-43 expression and hemichannel activity, but not gap junction communication in astrocyte cultures prepared from cortices and spinal cords. Spinal injection of TNF-α-activated astrocytes was sufficient to induce persistent mechanical allodynia, and this allodynia was suppressed by CXCL1 neutralization, CXCL1 receptor (CXCR2) antagonist, and pretreatment of astrocytes with connexin-43 small interfering RNA. Furthermore, nerve injury persistently increased excitatory synaptic transmission (spontaneous excitatory postsynaptic currents) in spinal lamina IIo nociceptive synapses in the late phase, and this increase was suppressed by carbenoxolone and Gap27, and recapitulated by CXCL1. Together, our findings demonstrate a novel mechanism of astrocytic connexin-43 to enhance spinal cord synaptic transmission and maintain neuropathic pain in the late-phase via releasing chemokines.
doi:10.1093/brain/awu140
PMCID: PMC4107738  PMID: 24919967
carbenoxolone (CBX); CXCL1; CXCR2; hemichannels; neuro-glial interaction
2.  A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief 
Cell  2014;157(6):1393-1404.
Summary
Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch, despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.
doi:10.1016/j.cell.2014.03.064
PMCID: PMC4098795  PMID: 24856969
3.  Extracellular microRNAs activate nociceptor neurons to elicit pain via TLR7 and TRPA1 
Neuron  2014;82(1):47-54.
SUMMARY
Intracellular microRNAs (miRNAs) are key regulators of gene expression. The role of extracellular miRNAs in neuronal activation and sensory behaviors are unknown. Here we report an unconventional role of extracellular miRNAs for rapid excitation of nociceptor neurons via toll-like receptor-7 (TLR7) and its coupling to TRPA1 ion channel. miRNA-let-7b induces rapid inward currents and action potentials in dorsal root ganglion (DRG) neurons. These responses require the GUUGUGU motif, only occur in neurons co-expressing TLR7 and TRPA1, and are abolished in mice lacking Tlr7 or Trpa1. Furthermore, let-7b induces TLR7/TRPA1-dependent single channel activities in DRG neurons and HEK293 cells over-expressing TLR7/TRPA1. Intraplantar injection of let-7b elicits rapid spontaneous pain via TLR7 and TRPA1. Finally, let-7b can be released from DRG neurons by neuronal activation, and let-7b inhibitor reduces formalin-induced TRPA1 currents and spontaneous pain. Thus, secreted extracellular miRNAs may serve as novelpain mediatorsvia activating TLR7 /TRPA1in nociceptor neurons.
doi:10.1016/j.neuron.2014.02.011
PMCID: PMC3982230  PMID: 24698267
DRG neurons; microRNAs; Toll-like receptor 7; TRPA1
4.  New insights into the mechanisms of itch: are pain and itch controlled by distinct mechanisms? 
Pflugers Archiv : European journal of physiology  2013;465(12):10.1007/s00424-013-1284-2.
Itch and pain are closely related but distinct sensations. They share largely overlapping mediators and receptors, and itch-responding neurons are also sensitive to pain stimuli. Itch-mediating primary sensory neurons are equipped with distinct receptors and ion channels for itch transduction, including Mas-related G protein-coupled receptors (Mrgprs), protease-activated receptors (PARs), histamine receptors, bile acid receptor (TGR5), toll-like receptors (TLRs), and transient receptor potential subfamily V1/A1 (TRPV1/A1). Recent progress has indicated the existence of an itch-specific neuronal circuitry. The MrgprA3-expressing primary sensory neurons exclusively innervate the epidermis of skin and their central axons connect with gastrin-releasing peptide receptor (GRPR)-expressing neurons in the superficial spinal cord. Notably, ablation of MrgprA3-expressing primary sensory neurons or GRPR-expressing spinal cord neurons results in selective reduction in itch but not pain. Chronic itch results from dysfunction of the immune and nervous system and can manifest as neural plasticity, despite the fact that chronic itch is often treated by dermatologists. While differences between acute pain and acute itch are striking, chronic itch and chronic pain share many similar mechanisms, including peripheral sensitization (increased responses of primary sensory neurons to itch and pain mediators), central sensitization (hyperactivity of spinal projection neurons and excitatory interneurons), loss of inhibitory control in the spinal cord, and neuro-immune and neuro-glial interactions. Notably, painful stimuli can elicit itch in some chronic conditions (e.g., atopic dermatitis) and some drugs for treating chronic pain are also effective in chronic itch. Thus, itch and pain have more similarities in pathological and chronic conditions.
doi:10.1007/s00424-013-1284-2
PMCID: PMC3796138  PMID: 23636773
Central sensitization; neuro-immune interaction; nociceptor; peripheral sensitization; pruritus; pruriceptor
5.  Emerging targets in neuroinflammation-driven chronic pain 
Nature reviews. Drug discovery  2014;13(7):533-548.
Current analgesics predominately modulate pain transduction and transmission in neurons and have limited success in controlling disease progression. Accumulating evidence suggests that neuroinflammation, which is characterized by infiltration of immune cells, activation of glial cells and production of inflammatory mediators in the peripheral and central nervous system, has an important role in the induction and maintenance of chronic pain. This review focuses on emerging targets such as chemokines, proteases and the Wnt pathway that promote spinal cord neuroinflammation and chronic pain. It also highlights the anti-inflammatory and pro-resolution lipid mediators that act on immune cells, glial cells and neurons to resolve neuroinflammation, synaptic plasticity and pain. Targeting excessive neuroinflammation could offer new therapeutic opportunities for chronic pain and related neurological and psychiatric disorders.
doi:10.1038/nrd4334
PMCID: PMC4228377  PMID: 24948120
6.  Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice 
Neuroscience  2013;247:376-385.
Accumulating evidence indicates that activation of spinal cord astrocytes contributes importantly to nerve injury and inflammation-induced persistent pain and chronic opioid-induced antinociceptive tolerance. Phosphorylation of extracellular signal-regulated kinase (pERK) and induction of interleukin-1 beta (IL-1β) in spinal astrocytes have been implicated in astrocytes-mediated pain. Tissue plasminogen activator (tPA) is a serine protease that has been extensively used to treat stroke. We examined the potential involvement of tPA in chronic opioid-induced antinociceptive tolerance and activation of spinal astrocytes using tPA knockout (tPA−/−) mice and astrocyte cultures. tPA−/− mice exhibited unaltered nociceptive pain and morphine-induced acute analgesia. However, the antinociceptive tolerance, induced by chronic morphine (10 mg/kg/day, s.c.), is abrogated in tPA−/− mice. Chronic morphine induces tPA expression in GFAP-expressing spinal cord astrocytes. Chronic morphine also increases IL-1β expression in GFAP-expressing astrocytes, which is abolished in tPA-deficient mice. In cultured astrocytes, morphine treatment increases tPA, IL-1β, and pERK expression, and the increased IL-1β and pERK expression is abolished in tPA-deficient astrocytes. tPA is also sufficient to induce IL-1β and pERK expression in astrocyte cultures. Intrathecal injection of tPA results in up-regulation of GFAP and pERK in spinal astrocytes but not up-regulation of IBA-1 in spinal microglia. Finally, intrathecal tPA elicits persistent mechanical allodynia, which is inhibited by the astroglial toxin alpha-amino adipate and the MEK (ERK kinase) inhibitor U0126. Collectively, these data suggest an important role of tPA in regulating astrocytic signaling, pain hypersensitivity, and morphine tolerance.
doi:10.1016/j.neuroscience.2013.05.018
PMCID: PMC3722295  PMID: 23707980
acute opioid analgesia; chronic morphine exposure; extracellular signal-regulated kinase (ERK); interleukin-1 beta (IL-1β); protease; tPA knockout mice
7.  Neuroprotectin/Protectin D1 protects neuropathic pain in mice after nerve trauma 
Annals of neurology  2013;74(3):490-495.
Prevalence of neuropathic pain is high after major surgeries. However, effective treatment for preventing neuropathic pain is lacking. Here we report that peri-surgical treatment of Neuroprotectin D1/protectin D1 (NPD1/PD1), derived from docosahexaenoic acid, prevents nerve injury-induced mechanical allodynia and ongoing pain in mice. Intrathecal post-treatment of NPD1/PD1 also effectively reduces established neuropathic pain and produces no apparent signs of analgesic tolerance. Mechanistically, NPD1/PD1 treatment blocks nerve injury-induced long-term potentiation, glial reaction, inflammatory responses, and reverses synaptic plasticity in the spinal cord. Thus, NPD1/PD1 and related mimetics might serve as a new class of analgesics for preventing and treating neuropathic pain.
doi:10.1002/ana.23928
PMCID: PMC3791159  PMID: 23686636
8.  Resolvin E1 inhibits neuropathic pain and spinal cord microglial activation following peripheral nerve injury 
Accumulating evidence indicates that activation of spinal cord microglia plays an important role in the genesis of neuropathic pain. Resolvin E1 (E1) is derived from omega-3 polyunsaturated fatty acid and exhibits potent anti-inflammatory, pro-resolution, and anti-nociceptive effects. We further examined whether RvE1 could reduce neuropathic pain and modulate spinal cord microglial activation. Intrathecal pre-treatment of RvE1 (100 ng) daily for 3 days partially prevented the development of nerve injury-induced mechanical allodynia and up-regulation of IBA-1 (microglial marker) and TNF-α in the spinal cord dorsal horn. Furthermore, intrathecal post-treatment of RvE1 (100 ng), 3 weeks after nerve injury, transiently reduced mechanical allodynia and heat hyperalgesia. Finally, RvE1 blocked lipopolisaccharide-induced microgliosis and TNF-α release in primary micoglial cultures. Our data suggest that RvE1 may attenuate neuropathic pain via inhibiting microglial signaling. Targeting the anti-inflammatory and pro-resolution lipid mediators may offer new options for preventing and treating neuropathic pain.
doi:10.1007/s11481-012-9394-8
PMCID: PMC3671904  PMID: 22878925
Eicosapentaenoic acid (EPA); Tumor necrosis factor-alpha TNF-α; lipopolisacride (LPS); nerve injury; omega-3 poly unsaturated fatty acids; spinal cord
9.  Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion 
The Journal of Clinical Investigation  2014;124(3):1173-1186.
Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α–induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.
doi:10.1172/JCI72230
PMCID: PMC3934175  PMID: 24531553
10.  Loss of NR1 Subunit of NMDARs in Primary Sensory Neurons Leads to Hyperexcitability and Pain Hypersensitivity: Involvement of Ca2+-Activated Small Conductance Potassium Channels 
The Journal of Neuroscience  2013;33(33):13425-13430.
It is well established that activation of NMDARs plays an essential role in spinal cord synaptic plasticity (i.e., central sensitization) and pain hypersensitivity after tissue injury. Despite prominent expression of NMDARs in DRG primary sensory neurons, the unique role of peripheral NMDARs in regulating intrinsic neuronal excitability and pain sensitivity is not well understood, in part due to the lack of selective molecular tools. To address this problem, we used Advillin-Cre driver to delete the NR1 subunit of NMDARs selectively in DRG neurons. In NR1 conditional knock-out (NR1-cKO) mice, NR1 expression is absent in DRG neurons but remains normal in spinal cord neurons; NMDA-induced currents are also eliminated in DRG neurons of these mice. Surprisingly, NR1-cKO mice displayed mechanical and thermal hypersensitivity compared with wild-type littermates. NR1-deficient DRG neurons show increased excitability, as indicated by increased frequency of action potentials, and enhanced excitatory synaptic transmission in spinal cord slices, as indicated by increased frequency of miniature EPSCs. This hyperexcitability can be reproduced by the NMDAR antagonist APV and by Ca2+-activated slow conductance K+ (SK) channel blocker apamin. Furthermore, NR1-positive DRG neurons coexpress SK1/SK2 and apamin-sensitive afterhyperpolarization currents are elevated by NMDA and suppressed by APV in these neurons. Our findings reveal the hitherto unsuspected role of NMDARs in controlling the intrinsic excitability of primary sensory neurons possibly via Ca2+-activated SK channels. Our results also call attention to potential opposing effects of NMDAR antagonists as a treatment for pain and other neurological disorders.
doi:10.1523/JNEUROSCI.0454-13.2013
PMCID: PMC3742928  PMID: 23946399
11.  Microglia and Spinal Cord Synaptic Plasticity in Persistent Pain 
Neural Plasticity  2013;2013:753656.
Microglia are regarded as macrophages in the central nervous system (CNS) and play an important role in neuroinflammation in the CNS. Microglial activation has been strongly implicated in neurodegeneration in the brain. Increasing evidence also suggests an important role of spinal cord microglia in the genesis of persistent pain, by releasing the proinflammatory cytokines tumor necrosis factor-alpha (TNFα), Interleukine-1beta (IL-1β), and brain derived neurotrophic factor (BDNF). In this review, we discuss the recent findings illustrating the importance of microglial mediators in regulating synaptic plasticity of the excitatory and inhibitory pain circuits in the spinal cord, leading to enhanced pain states. Insights into microglial-neuronal interactions in the spinal cord dorsal horn will not only further our understanding of neural plasticity but may also lead to novel therapeutics for chronic pain management.
doi:10.1155/2013/753656
PMCID: PMC3759269  PMID: 24024042
12.  TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice 
The Journal of Clinical Investigation  2012;122(6):2195-2207.
Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. TLRs mediate innate immunity and regulate neuropathic pain, but their roles in pruritus are elusive. Here, we report that scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Notably, we found that treatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3–/– mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3–/– mice but not Tlr7–/– mice. Consequently, central sensitization–driven pain hypersensitivity, but not acute pain, was impaired in Tlr3–/– mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3–/– mice. Our findings demonstrate a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization. TLR3 may serve as a new target for developing anti-itch treatment.
doi:10.1172/JCI45414
PMCID: PMC3366391  PMID: 22565312
13.  Glia and pain: Is chronic pain a gliopathy? 
Pain  2013;154(0 1):S10-S28.
Activation of glial cells and neuro-glial interactions are emerging as key mechanisms underlying chronic pain. Accumulating evidence has implicated 3 types of glial cells in the development and maintenance of chronic pain: microglia and astrocytes of the central nervous system (CNS), and satellite glial cells of the dorsal root and trigeminal ganglia. Painful syndromes are associated with different glial activation states: (1) glial reaction (ie, upregulation of glial markers such as IBA1 and glial fibrillary acidic protein (GFAP) and/or morphological changes, including hypertrophy, proliferation, and modifications of glial networks); (2) phosphorylation of mitogen-activated protein kinase signaling pathways; (3) upregulation of adenosine triphosphate and chemokine receptors and hemichannels and downregulation of glutamate transporters; and (4) synthesis and release of glial mediators (eg, cytokines, chemokines, growth factors, and proteases) to the extracellular space. Although widely detected in chronic pain resulting from nerve trauma, inflammation, cancer, and chemotherapy in rodents, and more recently, human immunodeficiency virus-associated neuropathy in human beings, glial reaction (activation state 1) is not thought to mediate pain sensitivity directly. Instead, activation states 2 to 4 have been demonstrated to enhance pain sensitivity via a number of synergistic neuro-glial interactions. Glial mediators have been shown to powerfully modulate excitatory and inhibitory synaptic transmission at presynaptic, postsynaptic, and extrasynaptic sites. Glial activation also occurs in acute pain conditions, and acute opioid treatment activates peripheral glia to mask opioid analgesia. Thus, chronic pain could be a result of “gliopathy,” that is, dysregulation of glial functions in the central and peripheral nervous system. In this review, we provide an update on recent advances and discuss remaining questions.
doi:10.1016/j.pain.2013.06.022
PMCID: PMC3858488  PMID: 23792284
Astrocytes; ATP receptors; Chemokines; Cytokines; Human; Microglia; Rodents; Satellite glial cells; Spinal cord
14.  Emerging Roles of Resolvins in the Resolution of Inflammation and Pain 
Trends in neurosciences  2011;34(11):599-609.
Resolvins, including D and E series resolvins, are endogenous lipid mediators generated during the resolution phase of acute inflammation from the omega-3 polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Resolvins are known to have potent anti-inflammatory and pro-resolution actions in several animal models of inflammation. Recent findings also demonstrate that resolvin E1 and resolvin D1 can each potently dampen inflammatory and postoperative pain. This review focuses on the mechanisms by which resolvins act on their receptors in immune cells and neurons to normalize exaggerated pain, via regulating inflammatory mediators, transient receptor potential (TRP) ion channels, and spinal cord synaptic transmission. Resolvins may offer novel therapeutic approaches for preventing and treating pain conditions associated with inflammation.
doi:10.1016/j.tins.2011.08.005
PMCID: PMC3200462  PMID: 21963090
15.  Resolvins are potent analgesics for arthritic pain 
British Journal of Pharmacology  2011;164(2):274-277.
Arthritis-associated inflammatory pain represents a serious medical problem worldwide. Current treatments for arthritic pain are not very effective and cause unwanted side effects. Resolvins, including the resolvin D and resolvin E series, are a family of novel endogenous lipid mediators derived from ω-3 polyunsaturated fatty acids and display potent anti-inflammatory and pro-resolution actions in animal models of inflammation. Emerging evidence also points to a potent anti-hyperalgesic role of resolvins in animal models of inflammatory pain. The study by Lima-Garcia et al. in this issue of the BJP demonstrated that systemic treatment with 17(R)-hydroxy-docosahexaenoic acid (17(R)HDoHE), the precursor of resolvin D series and its product, aspirin-triggered resolvin D1 (AT-RvD1), at very low doses (1 µg·kg−1), reduced inflammatory pain in an adjuvant-induced arthritis model. Particularly 17(R)HDoHE reduced joint stiffness but not paw and joint oedema. Given their potency and safety profile, resolvins may represent a new class of analgesics well suited to treating inflammatory pain associated with arthritis.
LINKED ARTICLE
This article is a commentary on Lima-Garcia et al., pp. 278–293 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2011.01345.x
doi:10.1111/j.1476-5381.2011.01348.x
PMCID: PMC3174408  PMID: 21418190
Resolvin; RvD1 precursor; lipid mediators; inflammatory pain; arthritis; dorsal root ganglion; spinal cord; TNF-α
16.  Emerging role of toll-like receptors in the control of pain and itch 
Neuroscience Bulletin  2012;28(2):131-144.
Toll-like receptors (TLRs) are germline-encoded pattern-recognition receptors (PRRs) to initiate innate immune responses by recognizing molecular structures shared by a wide range of pathogens, known as pathogen-associated molecular patterns (PAMPs). After tissue injury or cellular stress, TLRs can also detect endogenous ligands known as danger-associated molecular patterns (DAMPs). TLRs are expressed in various cell types in the central nervous system (CNS), including non-neuronal and neuronal cells, and contribute to both infectious and non-infectious disorders in the CNS. Following tissue insult and nerve injury, TLRs (such as TLR2, 3, and 4) induce the activation of microglia and astrocytes and the production of the proinflammatory cytokines in the spinal cord, leading to the development and maintenance of inflammatory pain and neuropathic pain. In particular, primary sensory neurons, such as nociceptors express TLRs (e.g., TLR4 and TLR7) to sense exogenous PAMPs and endogenous DAMPs released after tissue injury and cellular stress. These neuronal TLRs are new players in the processing of pain and itch by increasing the excitability of primary sensory neurons. Given the prevalence of chronic pain and itch and the suffering of the affected people, insights into TLR signaling in nervous system will open a new avenue for the management of clinical pain and itch.
PMCID: PMC3347759  PMID: 22466124
astrocytes; microglia; Toll-like receptor; Pain; itch; danger-associated molecular patterns (DAMPs); pathogen-associated molecular patterns (PAMPs)
17.  Oxidative stress induces itch via activation of transient receptor potential subtype ankyrin 1 (TRPA1) in mice 
Neuroscience Bulletin  2012;28(2):145-154.
Objective
To investigate the role of oxidative stress in itch-indicative scratching behavior in mice, and furthermore, to define the cellular and molecular mechanisms underlying oxidative stress-mediated itch.
Methods
Scratching behavior was induced by intradermal injection of oxidants, including hydrogen peroxide (H2O2) and tert-butylhydroperoxide (tBHP) into the nape of the neck in mice and observed for 30 min.
Results
Intradermal H2O2 (0.03-1%) or tert-butylhydroperoxide (tBHP, 1-30 μmol) elicited robust scratching behavior, displaying an inverted-U-shaped dose-related curve. Naloxone, an opioid receptor antagonist, but not morphine, largely suppressed the oxidants-induced scratching. Chlorpheniramine, a histamine H1 receptor antagonist, blocked histamine but not oxidants-induced scratching, indicating the involvement of histamine-independent mechanism in oxidants-evoked itch. Further, resiniferatoxin (RTX) treatment abolished oxidants-induced scratching, suggesting an essential role of C-fibers. Notably, blockade of transient receptor potential subtype ankyryn 1 (TRPA1) by selective TRPA1 antagonist HC-030031, or genetic deletion of Trpa1 but not Trpv1 resulted in a profound reduction in H2O2-evoked scratching. Finally, systemic administration of the antioxidants N-acetyl-L-cysteine (NAC) or trolox (a water-soluble vitamin E analogue) attenuated scratching induced by the oxidants.
Conclusion
Oxidative stress by different oxidants can induce profound scratching behavior, which is largely histamine and TRPV1-independent but TRPA1-dependent. Antioxidants and TRPA1 antagonists may be used to treat human itch conditions associated with oxidative stress.
PMCID: PMC3339413  PMID: 22466125
oxidative stress; anti-oxidants; itch; pruritus; TRPA1; TRPV1
18.  TNF-α contributes to spinal cord synaptic plasticity and inflammatory pain: Distinct role of TNF receptor subtype 1 and 2 
Pain  2010;152(2):419-427.
Tumor necrosis factor-alpha (TNF-α) is a key proinflammatory cytokine. It is generally believed that TNF-α exerts its effects primarily via TNF receptor subtype-1 (TNFR1). We investigated distinct role of TNFR1 and TNFR2 in spinal cord synaptic transmission and inflammatory pain. Compared to wild-type (WT) mice, TNFR1 and TNFR2 knockout (KO) mice exhibited normal heat sensitivity and unaltered excitatory synaptic transmission in the spinal cord, as revealed by spontaneous excitatory postsynaptic currents (sEPSCs) in lamina II neurons of spinal cord slices. However, heat hyperalgesia after intrathecal TNF-α and the second-phase spontaneous pain in the formalin test were reduced in both TNFR1- and TNFR2-KO mice. In particular, heat hyperalgesia after intraplantar injection of complete Freund's adjuvant (CFA) was decreased in the early phase in TNFR2-KO mice but reduced in both early and later phase in TNFR1-KO mice. Consistently, CFA elicited a transient increase of TNFR2 mRNA levels in the spinal cord on day 1. Notably, TNF-α evoked a drastic increase in sEPSC frequency in lamina II neurons, which was abolished in TNFR1-KO mice and reduced in TNFR2-KO mice. TNF-α also increased NMDA currents in lamina II neurons, and this increase was abolished in TNFR1-KO mice but retained in TNFR2-KO mice. Finally, intrathecal injection of the NMDA receptor antagonist MK-801 prevented heat hyperalgesia elicited by intrathecal TNF-α. Our findings support a central role of TNF-α in regulating synaptic plasticity (central sensitization) and inflammatory pain via both TNFR1 and TNFR2. Our data also uncover a unique role of TNFR2 in mediating early-phase inflammatory pain.
doi:10.1016/j.pain.2010.11.014
PMCID: PMC3022092  PMID: 21159431
proinflammatory cytokine; central sensitization; TNFR1; TNFR2; formalin; complete Freund's adjuvant
19.  Resolvin D2 is a potent endogenous inhibitor for transient receptor potential subtype V1/A1, inflammatory pain, and spinal cord synaptic plasticity in mice: Distinct roles of Resolvin D1, D2, and E1 
Inflammatory pain such as arthritic pain is typically treated with opioids and COX-2 inhibitors with well-known side effects. Transient receptor potential subtype V1 (TRPV1) and A1 (TRPA1) contribute importantly to the genesis of inflammatory pain via both peripheral mechanisms (peripheral sensitization) and spinal cord mechanisms (central sensitization). Although these TRP channels have been intensively studied, little is known about their endogenous inhibitors. Recent studies have demonstrated that the endogenous lipid mediators resolvins (RvE1 and RvD1), derived from omega-3 unsaturated fatty acids, are potent inhibitors for inflammatory pain, without noticeable side effects. However, the molecular mechanisms underlying resolvins’ distinct analgesic actions in mice are unclear. Resolvin D2 (RvD2) is a novel family member of resolvins. Here we report that RvD2 is a remarkably potent inhibitor of TRPV1 (IC50=0.1 nM) and TRPA1 (IC50= 2 nM) in primary sensory neurons, whereas RvE1 and RvD1 selectively inhibited TRPV1 (IC50=1 nM) and TRPA1 (IC50=9 nM), respectively. Accordingly, RvD2, RvE1, and RvD1 differentially regulated TRPV1 and TRPA1 agonist-elicited acute pain and spinal cord synaptic plasticity (sEPSC frequency increase). RvD2 also abolished inflammation-induced sEPSC increases (frequency and amplitude), without affecting basal synaptic transmission. Intrathecal administration of RvD2 at very low doses (0.01-1 ng) prevented formalin-induced spontaneous pain. Intrathecal RvD2 also reversed adjuvant-induced inflammatory pain without altering baseline pain and motor function. Finally, intrathecal RvD2 reversed C-fiber stimulation-evoked long-term potentiation in the spinal cord. Our findings suggest distinct roles of resolvins in regulating TRP channels and identify RvD2 as a potent endogenous inhibitor for TRPV1/A1 and inflammatory pain.
doi:10.1523/JNEUROSCI.4192-11.2011
PMCID: PMC3242808  PMID: 22171045
docosahexaenoic acid (DHA); eicosapentaenoic acid (EPA); long-term potentiation; omega-3 polyunsaturated fatty acid; primary sensory neurons; spontaneous EPSCs
20.  Spinal injection of TNF-α-activated astrocytes produces persistent pain symptom mechanical allodynia by releasing monocyte chemoattractant protein-1 
Glia  2010;58(15):1871-1880.
Accumulating evidence suggests that spinal astrocytes play an important role in the genesis of persistent pain, by increasing the activity of spinal cord nociceptive neurons, i.e., central sensitization. But direct evidence of whether activation of astrocytes is sufficient to induce chronic pain symptoms is lacking. We investigated whether and how spinal injection of activated astrocytes could produce mechanical allodynia, a cardinal feature of chronic pain, in naïve mice. Spinal (intrathecal) injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by tumor necrosis factor-alpha (TNF-α), induced a substantial decrease in paw withdrawal thresholds, indicating the development of mechanical allodynia. This allodynia was prevented when the astrocyte cultures were pre-treated with a peptide inhibitor of c-Jun N-terminal kinase (JNK), D-JNKI-1. Of note a short exposure of astrocytes to TNF-α for 15 minutes dramatically increased the expression and release of the chemokine monocyte chemoattractant protein-1 (MCP-1), even 3 hours after TNF-α withdrawal, in a JNK-dependent manner. In parallel, intrathecal administration of TNF-α induced MCP-1 expression in spinal cord astrocytes. In particular, mechanical allodynia induced by TNF-α-activated astrocytes was reversed by a MCP-1 neutralizing antibody. Finally, pretreatment of astrocytes with MCP-1 siRNA attenuated astrocytes-induced mechanical allodynia. Taken together, our results suggest that activated astrocytes are sufficient to produce persistent pain symptom in naïve mice by releasing MCP-1.
doi:10.1002/glia.21056
PMCID: PMC2951489  PMID: 20737477
TNF-α; MCP-1; JNK; astrocytes; central sensitization
21.  Resolving TRPV1 and TNF-α Mediated Spinal Cord Synaptic Plasticity and Inflammatory Pain with Neuroprotectin D1 
Mechanisms of inflammatory pain are not fully understood. We investigated the role of TRPV1 and TNF-α, two critical mediators for inflammatory pain, in regulating spinal cord synaptic transmission. We found in mice lacking Trpv1 the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in lamina II neurons of spinal cord slices is reduced. Further, C-fiber-induced spinal long-term potentiation (LTP) in vivo is abolished in Trpv1 knockout mice. TNF-α also increases sEPSC frequency but not amplitude in spinal lamina IIo neurons, and this increase is abolished in Trpv1 knockout mice. Single-cell PCR analysis revealed that TNF-α-responding neurons in lamina IIo are exclusively excitatory (vGluT2+) neurons. Notably, neuroprotectin-1 (NPD1), an anti-inflammatory lipid mediator derived from omega-3 polyunsaturated fatty acid (docosahexaenoic acid) blocks TNF-α- and capsaicin-evoked sEPSC frequency increases but has no effect on basal synaptic transmission. Strikingly, NPD1 potently inhibits capsaicin-induced TRPV1 current (IC50=0.4 nM) in dissociated dorsal root ganglion neurons, and this IC50 is ≈ 500 times lower than that of AMG9810, a commonly used TRPV1 antagonist. NPD1 inhibition of TRPV1 is mediated by GPCRs, since the effects were blocked by pertussis toxin. In contrast, NPD1 had not effect on mustard oil-induced TRPA1 currents. Spinal injection of NPD1, at very low doses (0.1–10 ng), blocks spinal LTP and reduces TRPV1-dependent inflammatory pain, without affecting baseline pain. NPD1 also reduces TRPV1-independent but TNF-α-dependent pain hypersensitivity. Our findings demonstrate a novel role of NPD1 in regulating TRPV1/TNF-α-mediated spinal synaptic plasticity and identify NPD1 as a novel analgesic for treating inflammatory pain.
doi:10.1523/JNEUROSCI.2443-11.2011
PMCID: PMC3202339  PMID: 22016541
central sensitization; docosahexaenoic acid; long-term potentiation; omega-3 polyunsaturated fatty acid; primary sensory neurons; single-cell PCR; spontaneous EPSCs; TRPA1
22.  Light touch induces ERK activation in superficial dorsal horn neurons after inflammation: involvement of spinal astrocytes and JNK signaling in touch-evoked central sensitization and mechanical allodynia 
Journal of neurochemistry  2010;115(2):505-514.
Activation of extracellular signal-regulated kinase (ERK) in spinal cord neurons could serve as a marker for sensitization of dorsal horn neurons in persistent pain. ERK is normally activated by high-threshold noxious stimuli. We investigated how low-threshold mechanical stimuli could activate ERK after complete Freund’s adjuvant (CFA)-induced inflammation. Unilateral injection of CFA induced ipsilateral heat hyperalgesia and bilateral mechanical allodynia. CFA-induced ERK activation in ipsilateral dorsal horn neurons declined after 2 days. Interestingly, low threshold mechanical stimulation given by light touch either on the inflamed paw or the contralateral non-inflamed paw dramatically increased ERK phosphorylation (pERK) in the dorsal horn ipsilateral to touch stimulation. Notably, light touch induced pERK mainly in superficial neurons in laminae I-IIo. Intrathecal administration of the astroglial toxin L-α-aminoadipate (L-α-AA) on post-CFA day 2 reversed CFA-induced bilateral mechanical allodynia but not heat hyperalgesia. Furthermore, L-α-AA, the glial inhibitor fluorocitrate, and a peptide inhibitor of c-Jun N-terminal Kinase (JNK) all reduced light touch-evoked ERK activation ipsilateral to touch. Collectively, these data suggest that (a) ERK can be activated in superficial dorsal horn neurons by low threshold mechanical stimulation under pathological condition and (b) ERK activation by light touch is associated with mechanical allodynia and requires an astrocyte network.
doi:10.1111/j.1471-4159.2010.06946.x
PMCID: PMC2970698  PMID: 20722971
ERK; Complete Freund’s adjuvant; Mechanical allodynia; Astrocytes; JNK
23.  Role of microglia in neuropathic pain, postoperative pain, and morphine tolerance 
Management of chronic pain such as nerve injury-induced neuropathic pain associated with diabetic neuropathy, viral infection, and cancer is a real clinical challenge. Major surgeries such as breast and thoracic surgery, leg amputation, and coronary artery bypass surgery also lead to chronic pain in 10–50% of individuals after acute postoperative pain, in part due to surgery-induced nerve injury. Current treatments mainly focus on blocking neurotransmission in the pain pathway and have only resulted in limited success. Ironically, chronic opioid exposure may lead to paradoxical pain. Development of effective therapeutic strategies requires a better understanding of cellular mechanisms underlying the pathogenesis of neuropathic pain. An important progress in pain research points to important role of microglial cells in the development of chronic pain. Spinal cord microglia are strongly activated after nerve injury, surgical incision, and chronic opioid exposure. Increasing evidence suggests that under all these conditions the activated microglia not only exhibit increased expression of microglial markers CD11b and Iba1 but also display elevated phosphorylation of p38 MAP kinase. Inhibition of spinal cord p38 has been shown to attenuate neuropathic pain and postoperative pain, as well as morphine-induced antinociceptive tolerance. Activation of p38 in spinal microglia results in increased synthesis and release of the neurotrophin BDNF and the proinflammatory cytokines IL-1β, IL-6, and TNF-α. These microglia-released mediators can powerfully modulate spinal cord synaptic transmission, leading to increased excitability of dorsal horn neurons, i.e. central sensitization, in part via suppressing inhibitory synaptic transmission. We review the studies that support the pronociceptive role of microglia in conditions of neuropathic pain, post-surgical pain, and opioid tolerance. Some of these studies have been accomplished by four Taiwanese anesthesiologists who are also co-authors of this review during their training at Harvard Medical School. We conclude that targeting microglial signalling may lead to more effective treatments for devastating chronic pain after diabetic neuropathy, viral infection, cancer, and major surgeries in part via improving the analgesic efficacy of opioids.
doi:10.1016/S0929-6646(11)60074-0
PMCID: PMC3169792  PMID: 21783017
Central sensitization; neuronal-glial interactions; proinflammatory cytokines; p38 MAP kinase; spinal cord
24.  Toll-like Receptor-7 Mediates Pruritus 
Nature neuroscience  2010;13(12):1460-1462.
Toll-like receptors (TLRs) are typically expressed in immune cells to regulate innate immunity. Here we report that functional TLR7 is expressed in C-fiber primary sensory neurons and important for inducing itch (pruritis) but not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Thus, we have uncovered TLR7 as a novel itch mediator and a potential therapeutic target for anti-itch treatment in skin disease conditions.
doi:10.1038/nn.2683
PMCID: PMC2991508  PMID: 21037581
25.  Resolvins RvE1 and RvD1Attenuate Inflammatory Pain via Central and Peripheral Actions 
Nature medicine  2010;16(5):592-597.
Inflammatory pain, such as arthritis pain, is a growing health problem1. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a novel family of lipid mediators including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, show remarkable potency in treating disease conditions associated with inflammation2, 3. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 (0.3–20 ng) potently reduces inflammatory pain behaviors in mice induced by intraplantar injection of formalin, carrageenan or complete Freund’s adjuvant, without affecting basal pain perception. Intrathecal RvE1 also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and TNF-α. RvE1 plays anti-inflammatory roles via reducing neutrophil infiltration, paw edema, and proinflammatory cytokine expression. RvE1 also abolishes TRPV1- and TNF-α-induced excitatory postsynaptic current increase and TNF-α-evoked NMDA receptor hyperactivity in spinal dorsal horn neurons, via inhibition of ERK signaling pathway. Thus, we demonstrate a novel role of resolvins in normalizing spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the remarkable potency of resolvins and well known side effects of opioids and COX inhibitors, resolvins may represent novel analgesics for treating inflammatory pain.
doi:10.1038/nm.2123
PMCID: PMC2866054  PMID: 20383154

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