Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Activation of extracellular signal-regulated protein kinases 5 in primary afferent neurons contributes to heat and cold hyperalgesia after inflammation

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.     

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.     

Effects of the neurotrophic factor artemin on sensory afferent development and sensitivity

Artemin is a neuronal survival and differentiation factor in the glial cell line-derived neurotrophic factor family.Its receptor GFRα3 is expressed by a subpopulation of nociceptor type sensory neurons in the dorsal root and trigeminal ganglia(DRG and TG).These neurons co-express the heat,capsaicin and proton-sensitive channel TRPV1 and the cold and chemical-sensitive channel TRPA1.To further investigate the effects of artemin on sensory neurons,we isolated transgenic mice(ARTN-OE mice) that overexpress art...     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.     

The Contribution of TRPM8 and TRPA1 Channels to Cold Allodynia and Neuropathic Pain

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.     

Peripheral inflammation increases Scya2 expression in sensory ganglia and cytokine and endothelial related gene expression in inflamed tissue

The sensation of pain (nociception) is a critical factor in host defense during tissue injury and inflammation and is initiated at the site of injury by activation of primary afferent C-fiber and A-∂ nerve endings. Inflammation induces tissue alterations that sensitize these nociceptive nerve terminals, contributing to persistent pain. To understand this 'algesic tissue environment' and peripheral nervous signaling to the CNS and immune system, we examined cytokine and endothelial-related gene expression profiles in inflamed rat tissues and corresponding dorsal root ganglia (DRG) by microarray and RT-PCR following hind paw injection of carrageenan. In inflamed tissue, forty-two cytokine and endothelial-related genes exhibited elevated expression. In contrast, in DRG, only Scya2 (chemokine C-C motif ligand 2) mRNA was up-regulated, leading to an increase in its gene product monocyte chemoattractant protein-1. Scya2 mRNA was localized by in situ hybridization-immunocytochemical double-labeling to a subpopulation of vanilloid receptor-1 (transient receptor potential vanilloid subtype 1) containing neurons, and its expression was increased by direct transient receptor potential vanilloid subtype 1 stimulation with the vanilloid agonist resiniferatoxin, indicating sensitivity to nociceptive afferent activity. Our results are consistent with the idea that monocyte chemoattractant protein-1 at the site of peripheral injury and/or in DRG is involved in inflammatory hyperalgesia.     

Experimental Trypanosoma cruzi infection in platelet-activating factor receptor-deficient mice

The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR(-/-) mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR(-/-) mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-gamma and TNF-alpha in heart tissue of infected animals. Moreover, serum concentrations of TNF-alpha, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR(-/-) animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B(4) or MCP-1 and produced less nitric oxide when infected and activated with IFN-gamma. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.     

Selective Inflammatory Pain Insensitivity in the African Naked Mole-Rat (Heterocephalus glaber)

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception.     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.     

Speaking out of turn: a role for silent synapses in pain

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.     

Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.     

Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice

Previous studies suggest that adenosine A₁ receptors (A₁R) modulate the processing of pain. The aim of this study was to characterize the distribution of A₁R in nociceptive tissues and to evaluate whether targeting A₁R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A₁R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A₁R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A₁R agonist. Despite expression of A₁R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A₁R agonists might be a valuable approach for the treatment of neuropathic pain.     

Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber)

In all mammals, tissue inflammation leads to pain and behavioral sensitization to thermal and mechanical stimuli called hyperalgesia. We studied pain mechanisms in the African naked mole-rat, an unusual rodent species that lacks pain-related neuropeptides (e.g., substance P) in cutaneous sensory fibers. Naked mole-rats show a unique and remarkable lack of pain-related behaviors to two potent algogens, acid and capsaicin. Furthermore, when exposed to inflammatory insults or known mediators, naked mole-rats do not display thermal hyperalgesia. In contrast, naked mole-rats do display nocifensive behaviors in the formalin test and show mechanical hyperalgesia after inflammation. Using electrophysiology, we showed that primary afferent nociceptors in naked mole-rats are insensitive to acid stimuli, consistent with the animal's lack of acid-induced behavior. Acid transduction by sensory neurons is observed in birds, amphibians, and fish, which suggests that this tranduction mechanism has been selectively disabled in the naked mole-rat in the course of its evolution. In contrast, nociceptors do respond vigorously to capsaicin, and we also show that sensory neurons express a transient receptor potential vanilloid channel-1 ion channel that is capsaicin sensitive. Nevertheless, the activation of capsaicin-sensitive sensory neurons in naked mole-rats does not produce pain-related behavior. We show that capsaicin-sensitive nociceptors in the naked mole-rat are functionally connected to superficial dorsal horn neurons as in mice. However, the same nociceptors are also functionally connected to deep dorsal horn neurons, a connectivity that is rare in mice. The pain biology of the naked mole-rat is unique among mammals, thus the study of pain mechanisms in this unusual species can provide major insights into what constitutes “normal” mammalian nociception.     

Plasticity and emerging role of BKCa channels in nociceptive control in neuropathic pain

Large‐conductance Ca²⁺‐activated K⁺ (BK_Ca, MaxiK) channels are important for the regulation of neuronal excitability. Peripheral nerve injury causes plasticity of primary afferent neurons and spinal dorsal horn neurons, leading to central sensitization and neuropathic pain. However, little is known about changes in the BK_Ca channels in the dorsal root ganglion (DRG) and spinal dorsal horn and their role in the control of nociception in neuropathic pain. Here we show that L5 and L6 spinal nerve ligation in rats resulted in a substantial reduction in both the mRNA and protein levels of BK_Ca channels in the DRG but not in the spinal cord. Nerve injury primarily reduced the BK_Ca channel immunoreactivity in small‐ and medium‐sized DRG neurons. Furthermore, although the BK_Ca channel immunoreactivity was decreased in the lateral dorsal horn, there was an increase in the BK_Ca channel immunoreactivity present on dorsal horn neurons near the dorsal root entry zone. Blocking the BK_Ca channel with iberiotoxin at the spinal level significantly reduced the mechanical nociceptive withdrawal threshold in control and nerve‐injured rats. Intrathecal injection of the BK_Ca channel opener [1,3‐dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one] dose dependently reversed allodynia and hyperalgesia in nerve‐ligated rats but it had no significant effect on nociception in control rats. Our study provides novel information that nerve injury suppresses BK_Ca channel expression in the DRG and induces a redistribution of BK_Ca channels in the spinal dorsal horn. BK_Ca channels are increasingly involved in the control of sensory input in neuropathic pain and may represent a new target for neuropathic pain treatment.     

State-dependent phosphorylation of epsilon-isozyme of protein kinase C in adult rat dorsal root ganglia after inflammation and nerve injury

The epsilon-isozyme of protein kinase C (PKCepsilon) and the vanilloid receptor 1 (VR1) are both expressed in dorsal root ganglion (DRG) neurons and are reported to be predominantly and specifically involved in nociceptive function. Using phosphospecific antibody against the C-terminal hydrophobic site Ser729 of PKCepsilon as a marker of enzyme activation, the state-dependent activation of PKCepsilon, as well as the expression of VR1 in rat DRG neurons, was evaluated in different experimental pain models in vivo. Quantitative analysis showed that phosphorylation of PKCepsilon in DRG neurons was significantly up-regulated after carrageen- and Complete Freund's Adjuvant-induced inflammation, while it was markedly down-regulated after chronic constriction injury. A double-labeling study showed that phosphorylation of PKCepsilon was expressed predominantly in VR1 immunoreactivity positive small diameter DRG neurons mediating the nociceptive information from peripheral tissue to spinal cord. The VR1 protein expression showed no significant changes after either inflammation or chronic constriction injury. These data indicate that functional activation of PKCepsilon has a close relationship with the production of inflammatory hyperalgesia and the sensitization of the nociceptors. Inflammatory mediator-induced activation of PKCepsilon and subsequent sensitization of VR1 to noxious stimuli by PKCepsilon may be involved in nociceptor sensitization.