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.
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.
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.
astrocytes; microglia; Toll-like receptor; Pain; itch; danger-associated molecular patterns (DAMPs); pathogen-associated molecular patterns (PAMPs)
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.
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.
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.
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.
oxidative stress; anti-oxidants; itch; pruritus; TRPA1; TRPV1
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.
proinflammatory cytokine; central sensitization; TNFR1; TNFR2; formalin; complete Freund's adjuvant
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.
TNF-α; MCP-1; JNK; astrocytes; central sensitization
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.
ERK; Complete Freund’s adjuvant; Mechanical allodynia; Astrocytes; JNK
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.
Clinical management of chronic pain after nerve injury (neuropathic pain) and tumor invasion (cancer pain) is a real challenge due to our limited understanding of the cellular mechanisms that initiate and maintain chronic pain. It has been increasingly recognized that glial cells, such as microglia and astrocytes in the central nervous system play an important role in the development and maintenance of chronic pain. Notably, astrocytes make very close contacts with synapses and astrocyte reaction after nerve injury, arthritis, and tumor growth is more persistent than microglial reaction and displays a better correlation with chronic pain behaviors. Accumulating evidence indicates that activated astrocytes can release proinflammatory cytokines (e.g., IL-1β) and chemokines (e.g., MCP-1/CCL2) in the spinal cord to enhance and prolong persistent pain states. IL-1β can powerfully modulate synaptic transmission in the spinal cord by enhancing excitatory synaptic transmission and suppressing inhibitory synaptic transmission. IL-1β activation (cleavage) in the spinal cord after nerve injury requires the matrix metalloprotease-2 (MMP-2). In particular, nerve injury and inflammation activate the c-Jun N-terminal kinase (JNK) in spinal astrocytes, leading to a substantial increase in the expression and release of MCP-1. MCP-1 increases pain sensitivity via direct activation of NMDA receptors in dorsal horn neurons. Pharmacological inhibition of the IL-1β, JNK, MCP-1, or MMP-2 signaling via spinal administration has been shown to attenuate inflammatory, neuropathic, or cancer pain. Therefore, interventions in specific signaling pathways in astrocytes may offer new approaches for the management of chronic pain.
Neuropathic pain; nerve injury; spinal cord; cytokine; chemokine; MAP kinase; glia
Millions of people worldwide suffer from neuropathic pain as a result of damage to or dysfunction of the nervous system under various disease conditions. Development of effective therapeutic strategies requires a better understanding of molecular and cellular mechanisms underlying the pathogenesis of neuropathic pain. It has been increasingly recognized that spinal cord glial cells such as microglia and astrocytes play a critical role in the induction and maintenance of neuropathic pain by releasing powerful neuromodulators such as proinflammatory cytokines and chemokines. Recent evidence reveals chemokines as new players in pain control. In this article, we review evidence for chemokine modulation of pain via neuronal-glial interactions by focusing on the central role of two chemokines, CX3CL1 (fractalkine) and CCL2 (MCP-1), because they differentially regulate neuronal-glial interactions. Release of CX3CL1 from neurons is ideal to mediate neuronal-to-microglial signaling, since the sole receptor of this chemokine, CX3CR1, is expressed in spinal microglia and activation of the receptor leads to phosphorylation of p38 MAP kinase in microglia. Although CCL2 was implicated in neuronal-to-microglial signaling, a recent study shows a novel role of CCL2 in astroglial-to-neuronal signaling after nerve injury. In particular, CCL2 rapidly induces central sensitization by increasing the activity of NMDA receptors in dorsal horn neurons. Insights into the role of chemokines in neuronal-glial interactions after nerve injury will identify new targets for therapeutic intervention of neuropathic pain.
CCL2/MCP-1; CX3CL1/fractalkine; astrocytes; microglia; nerve injury; spinal cord
Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity via non-transcriptional and transcriptional regulation. In particular, ERK activation in spinal cord dorsal horn neurons by nociceptive activity, via multiple neurotransmitter receptors, and using different second messenger pathways plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. ERK activation in amygdala neurons is also required for inflammatory pain sensitization. After nerve injury, ERK, p38, and JNK are differentially activated in spinal glial cells (microglia vs astrocytes), leading to the synthesis of proinflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models. Development of specific inhibitors for MAPK pathways to target neurons and glial cells may lead to new therapies for pain management. Although it is well documented that MAPK pathways can increase pain sensitivity via peripheral mechanisms, this review will focus on central mechanisms of MAPKs, especially ERK.
MAPK; neural plasticity; central sensitization; spinal cord; amygdala; microglia; astrocytes; inflammatory pain; neuropathic pain
c-Fos, the protein of the protooncogene c-fos, has been extensively used as a marker for the activation of nociceptive neurons in the spinal cord for more than twenty years since Hunt et al. first reported that peripheral noxious stimulation to a hind paw of rats leads to a marked induction of c-Fos in superficial and deep dorsal horn neurons in 1987. In 1999, Ji et al. reported that phosphorylated extracellular signal-regulated kinase (pERK) is specifically induced by noxious stimulation in superficial dorsal horn neurons. Accumulating evidence indicates that pERK induction or ERK activation in dorsal horn neurons is essential for the development of central sensitization, increased sensitivity of dorsal horn neurons that is responsible for the generation of persistent pain. Further, molecular mechanisms underlying ERK-mediated central sensitization have been revealed. In contrast, direct evidence for c-Fos-mediated central sensitization is not sufficient. After a noxious stimulus (e.g., capsaicin injection) or tissue injury, c-Fos begins to be induced after 30-60 minutes, whereas pERK can be induced within a minute, which can correlate well with the development of pain hypersensitivity. While c-Fos is often induced in the nuclei of neurons, pERK can be induced in different subcellular structures of neurons such as nuclei, cytoplasma, axons, and dendrites. pERK can even be induced in spinal cord microglia and astrocytes after nerve injury. In summary, both c-Fos and pERK can be used as markers for neuronal activation following noxious stimulation and tissue injury, but pERK is much more dynamic and appears to be a better marker for central sensitization.
Neuropathic pain affects millions of people globally and could be a disease on its own right. Current treatments focus on blocking neurotransmission and have resulted in limited success. Recent progress points to an important role of neuroinflammation in the pathogenesis of neuropathic pain. Matrix metalloproteases (MMPs) comprise a large family of zinc endopeptidases that have been implicated in the generation of neuroinflammation via cleavage of extracellular matrix proteins and activation of proinflammatory cytokines and chemokines. However, little is known about the role of MMPs in chronic pain regulation. Our recent study has shown that neuropathic pain development in the early and late phase requires MMP-9 and MMP-2, respectively. Inhibition of MMP-9 or MMP-2 may provide a new strategy for the prevention and treatment of neuropathic pain.
The c-Jun N-terminal kinase (JNK) is a stress-activated member of MAP kinase family. JNK activation has been strongly implicated in inflammatory responses, neurodegeneration, and apoptosis. Recent evidence shows that JNK pathway is also transiently activated in primary sensory neurons after tissue or nerve injury, which is required for the development of hyperalgesia and allodynia. In particular, JNK is persistently activated in astrocytes of the spinal cord after nerve injury, and this activation can maintain central sensitization and mechanical allodynia. In this mini-review, we will provide evidence for the involvement of JNK pathway in regulating persistent pain sensitization. We will also discuss possible upstream signaling mechanisms that cause JNK activation and downstream signaling mechanisms by which JNK modulates pain sensitivity. Thus, targeting JNK pathway might be a useful strategy to treat both neurodegeneration and chronic pain.
During evolution, living organisms develop a specialized apparatus called nociceptors to sense their environment and avoid hazardous situations. Intense stimulation of high threshold C- and Aδ-fibers of nociceptive primary sensory neurons will elicit pain, which is acute and protective under normal conditions. A further evolution of the early pain system results in the development of nociceptor sensitization under injury or disease conditions, leading to enhanced pain states. This sensitization in the peripheral nervous system is also called peripheral sensitization, as compared to its counterpart, central sensitization. Inflammatory mediators such as proinflammatory cytokines (TNF-α, IL-1β), PGE2, bradykinin, and NGF increase the sensitivity and excitability of nociceptors by enhancing the activity of pronociceptive receptors and ion channels (e.g., TRPV1 and Nav1.8). We will review the evidence demonstrating that activation of multiple intracellular signal pathways such as MAPK pathways in primary sensory neurons results in the induction and maintenance of peripheral sensitization and produces persistent pain. Targeting the critical signaling pathways in the periphery will tackle pain at the source.
Peripheral sensitization; dorsal root ganglion; inflammatory pain; neuropathic pain; MAP kinases; neural plasticity
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 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.
Chemokine; MAP kinase; microglia; spinal cord; neural-glial interaction; spinal nerve ligation; intracellular signaling; neuropathic pain
Thoracotomy often results in chronic pain, characterized by resting pain and elevated mechano-sensitivity. This paper defines complex behavioral responses to tactile stimulation in rats after thoracotomy, shown to be reversibly relieved by systemic morphine, in order to develop a novel qualitative “pain” score. A deep incision and 1 hour of rib retraction in male Sprague-Dawley rats resulted in reduced threshold and a change in the locus of greatest tactile (von Frey filament) sensitivity, from the lower back to a more rostral location around the wound site, and extending bilaterally. The fraction of rats showing nocifensive responses to mild stimulation (10 gm) increased after thoracotomy (from a pre-operative value of 0/10 to 8/10 at 10 days post-op), and the average threshold decreased correspondingly, from 15 gm to ∼4 gm. The nature of the nocifensive responses to tactile stimulation, composed pre-operatively only of no response (Grade 0) or brief contractions of the local subcutaneous muscles (Grade I), changed markedly after thoracotomy, with the appearance of new behaviors including a brisk lateral “escape” movement and/or a 180° rotation of the trunk (both included as Grade II), and whole body shuddering, and scratching and squealing (Grade III). Systemic morphine (2.5 mg/kg, i.p.) transiently raised the threshold for response and reduced the frequency of Grade II and III responses, supporting the interpretation that these represent pain. The findings support the development of a Qualitative Hyperalgesic Profile to assess the complex behavior that indicates a central integration of hyperalgesia.
Post-operative; allodynia; morphine; mechano-sensitivity; preventive analgesia
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.
Recent studies have indicated an important role of chemokines such as CCL2 in the development of chronic pain. However, the distinct roles of different chemokines in the development and maintenance of neuropathic pain and in their interactions with neurons have not been clearly elucidated. We found that spinal nerve ligation (SNL) not only induced persistent neuropathic pain symptoms including mechanical allodynia and heat hyperalgesia but also produced sustained CXCL1 upregulation in the spinal cord. Double staining of immunofluorescence and in situ hybridization revealed that CXCL1 was primarily induced in spinal astrocytes. In cultured astrocytes, TNF-α induced robust CXCL1 expression, via the activation of the c-jun N-terminal kinase (JNK). Intrathecal administration of CXCL1 neutralizing antibody transiently reduced SNL-induced pain hypersensitivity, suggesting an essential role of CXCL1 in neuropathic pain sensitization. In particular, intraspinal delivery of CXCL1 shRNA lentiviral vectors, either before or after SNL, persistently attenuated SNL-induced pain hypersensitivity. Spinal application of CXCL1 not only elicited pain hypersensitivity but also induced rapid neuronal activation, as indicated by the expression of phosphorylated ERK and CREB (pERK and pCREB) and c-Fos in spinal cord neurons. Interestingly, CXCR2, the primary receptor of CXCL1, was upregulated in dorsal horn neurons after SNL, and the CXCR2 antagonist SB225002 completely blocked the CXCL1-induced heat hyperalgesia. SB225002 also attenuated SNL-induced pain hypersensitivity. Collectively, our results have demonstrated a novel form of chemokine-mediated glial-neuronal interaction in the spinal cord that can drive neuropathic pain. Inhibition of the CXCL1-CXCR2 signaling may offer a new therapy for neuropathic pain management.
Chronic neuropathic pain is a frequent consequence of spinal cord injury (SCI). Yet despite recent advances, up-stream releasing mechanisms and effective therapeutic options remain elusive. Previous studies have demonstrated that SCI results in excessive ATP release to the peri-traumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. We sought to assess the role of connexin 43 (Cx43) as a mediator of CNS inflammation and chronic pain.
To determine the extent of Cx43 involvement in chronic pain, a weight-drop SCI was performed on transgenic mice with Cx43/Cx30 deletions. SCI induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after 4 weeks and was maintained after 8 weeks. Notably, SCI-induced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with Cx43/Cx30 deletions, but fully developed in transgenic mice with only Cx30 deletion. SCI-induced gliosis, detected as upregulation of glial-fibrillary-acidic- protein (GFAP) in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with Cx43/Cx30 deletions, when compared to littermate controls. In comparison, a standard regimen of post-SCI treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than Cx43 deletion.
These findings suggest Cx43 is critically linked to the development of central neuropathic pain following acute SCI. Since Cx43/Cx30 is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain.
Astrocyte; ATP; chronic pain; Connexin 43; P2X; spinal cord injury
Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.
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.
Despite decades of intense research efforts, actions of acute opioids are not fully understood. Increasing evidence suggests that in addition to well-documented antinociceptive effects opioids also produce paradoxical hyperalgesic and excitatory effects on neurons. However, most studies focus on the pronociceptive actions of chronic opioid exposure. Matrix metalloproteinase 9 (MMP-9) plays an important role in neuroinflammation and neuropathic pain development. We examined MMP-9 expression and localization in dorsal root ganglia (DRGs) after acute morphine treatment and, furthermore, the role of MMP-9 in modulating acute morphine-induced analgesia and hyperalgesia in mice.
Subcutaneous morphine induced a marked up-regulation of MMP-9 protein in DRGs but not spinal cords. Morphine also increased MMP-9 activity and mRNA expression in DRGs. MMP-9 up-regulation peaked at 2 h but returned to the baseline after 24 h. In DRG tissue sections, MMP-9 is expressed in small and medium-sized neurons that co-express mu opioid receptors (MOR). In DRG cultures, MOR agonists morphine, DAMGO, and remifentanil each increased MMP-9 expression in neurons, whereas the opioid receptor antagonist naloxone and the MOR-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) suppressed morphine-induced MMP-9 expression. Notably, subcutaneous morphine-induced analgesia was enhanced and prolonged in Mmp9 knockout mice and also potentiated in wild-type mice receiving intrathecal injection of MMP-9 inhibitors. Consistently, intrathecal injection of specific siRNA targeting MMP-9 reduced MMP-9 expression in DRGs and enhanced and prolonged morphine analgesia. Subcutaneous morphine also produced heat hyperalgesia at 24 h, but this opioid-induced hyperalgesia was not enhanced after MMP-9 deletion or inhibition.
Transient MMP-9 up-regulation in DRG neurons can mask opioid analgesia, without modulating opioid-induced hyperalgesia. Distinct molecular mechanisms (MMP-9 dependent and independent) control acute opioid-induced pronociceptive actions (anti-analgesia in the first several hours and hyperalgesia after 24 h). Targeting MMP-9 may improve acute opioid analgesia.
Dorsal root ganglion; Metalloprotease; MMP-9; mu opioid receptor (MOR); Opioid-induced analgesia; Opioid-induced hyperalgesia (OIH); Spinal cord
Activation of spinal cord glial cells such as microglia and astrocytes has been shown to regulate chronic opioid-induced antinociceptive tolerance and hyperalgesia, due to spinal up-regulation of the proinflammatory cytokines such as interleukin-1 beta (IL-1β). Matrix metalloprotease-9 (MMP-9) has been implicated in IL-1β activation in neuropathic pain. However, it is unclear whether acute opioid treatment can activate glial cells in the peripheral nervous system. We examined acute morphine-induced activation of satellite glial cells (SGCs) and up-regulation of IL-1β in dorsal root ganglia (DRGs), and further investigated the involvement of MMP-9 in these opioid-induced peripheral changes.
Subcutaneous morphine injection (10 mg/kg) induced robust peripheral glial responses, as evidenced by increased GFAP expression in DRGs but not in spinal cords. The acute morphine-induced GFAP expression is transient, peaking at 2 h and declining after 3 h. Acute morphine treatment also increased IL-1β immunoreactivity in SGCs and IL-1β activation in DRGs. MMP-9 and GFAP are expressed in DRG neurons and SGCs, respectively. Confocal analysis revealed a close proximity of MMP-9 and GFAP immunostaining. Importantly, morphine-induced DRG up-regulation of GFAP expression and IL-1β activation was abolished after Mmp9 deletion or naloxone pre-treatment. Finally, intrathecal injections of IL-1β-selective siRNA not only reduced DRG IL-1β expression but also prolonged acute morphine-induced analgesia.
Acute morphine induces opioid receptors- and MMP-9-dependent up-regulation of GFAP expression and IL-1β activation in SGCs of DRGs. MMP-9 could mask and shorten morphine analgesia via peripheral neuron-glial interactions. Targeting peripheral glial activation might prolong acute opioid analgesia.