[Show abstract][Hide abstract] ABSTRACT: Artemin, a member of the glial cell line-derived neurotrophic factor family, is known to have a variety of neuronal functions, and has been the subject of attention because it has interesting effects, including bi-directional results in modulation in neuropathic and inflammatory pain. It has been shown that the overexpression of artemin is associated with an increase in the expression of TRP family channels in primary afferents and subsequent hyperalgesia, and an increase in neuronal activity. The purpose of this study was to examine the peripheral synthesis of artemin in inflammatory and neuropathic pain models, and to demonstrate the effects of long-term or repeated application of artemin in vivo on pain behaviors and on the expression of TRP family channels. Further, the regulatory mechanisms of artemin on TRPV1/A1 were examined using cultured DRG neurons.
We have demonstrated that artemin is locally elevated in skin over long periods of time, that artemin signals significantly increase in deep layers of the epidermis, and also that it is distributed over a broad area of the dermis. In contrast, NGF showed transient increases after peripheral inflammation. It was confirmed that the co-localization of TRPV1/A1 and GFRα3 was higher than that between TRPV1/A1 and TrkA. In the peripheral sciatic nerve trunk, the synthesis of artemin was found by RT-PCR and in situ hybridization to increase at a site distal to a nerve injury. We demonstrated that in vivo repeated artemin injections into the periphery changed the gene expression of TRPV1/A1 in DRG neurons without affecting GFRα3 expression. Repeated artemin injections also induced mechanical and heat hyperalgesia. Using primary cultured DRG neurons, we found that artemin application significantly increased TRPV1/A1 expression and Ca(2+) influx. Artemin-induced p38 MAPK pathway regulated the TRPV1 channel expression, however TRPA1 upregulation by artemin is not mediated through p38 MAPK.
These data indicate the important roles of peripherally-derived artemin on the regulation of TRPV1/A1 in DRG neurons in pathological conditions such as inflammatory and neuropathic pain.
[Show abstract][Hide abstract] ABSTRACT: We reported differential expression of the transient receptor potential vanilloid 1 (TRPV1), the transient receptor potential ankyrin 1 (TRPA1), and the (TRPM8) in the geniculate ganglions (GGs) of naive rats. In medical practice, the chorda tympani nerve (CTN) is injured in some patients during middle-ear surgery, and results in tongue numbness and taste disorder. We investigated changes in the expression of these receptors in GGs after CTN injury. In naive-rat GGs, 11.4, 11.8, and 0.5% of neurons were found to express the TRPV1, the TRPA1, the TRPM8, respectively. At 3 days after CTN injury, 5.2 and 4.0% of activating transcription factor 3-immunoreactive neurons, considered as injured neurons, were found to express the TRPV1 and the TRPA1, respectively. Among activating transcription factor 3-immunonegative neurons, considered as uninjured neurons, 3.9 and 3.8% were found to express the TRPV1 and the TRPA1, respectively. The TRPM8 was not detected in GGs after CTN injury. We found decreased mRNA levels of the TRPV1 and the TRPA1 in all neurons, as well as in uninjured neurons of ipsilateral GGs after CTN injury. CTN injury changes the gene expression in GGs and may have effects on the tongue.
[Show abstract][Hide abstract] ABSTRACT: Evodiae fructus (EF), a fruit of Evodia rutaecarpa Bentham, has long been used as an analgesic drug in traditional Chinese and Japanese medicine. However, the underlying molecular mechanism of its pharmacological action is unclear. Here, using calcium imaging, whole-cell patch-clamp recording, and behavioral analysis, we investigated the pharmacological action of EF and its principal compound, evodiamine, on the transient receptor potential (TRP) V1 channels. Dorsal root ganglion (DRG) neurons and TRPV1- or TRPA1-transfected human embryonic kidney-derived (HEK) 293 cells were used for calcium imaging or whole-cell patch-clamp recording. Twenty male adult Sprague-Dawley rats were used for the capsaicin-induced thermal hyperalgesia behavioral analyses. We found that evodiamine induced significant increases in intracellular calcium and robust inward currents in a subpopulation of isolated rat DRG neurons, most of which were also sensitive to capsaicin. The effect of evodiamine was completely blocked by capsazepine, a competitive antagonist of TRPV1. Evodiamine induced significant inward currents in TRPV1-, but not TRPA1-transfected HEK293 cells. Pretreatment with evodiamine reduced capsaicin-induced currents significantly. Furthermore, the in vivo pre-treatment of evodiamine suppressed thermal hyperalgesia induced by intraplantar injection of capsaicin in rats. These results identify that the analgesic effect of EF and evodiamine may be due to the activation and subsequent desensitization of TRPV1 in sensory neurons.
Journal of Natural Medicines 07/2015; DOI:10.1007/s11418-015-0929-1 · 1.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have indicated an important role of ATP receptors in spinal microglia, such as P2Y12 or P2Y13, in the development of chronic pain. However, intracellular signaling cascade of these receptors have not been clearly elucidated. We found that intrathecal injection of 2-(methylthio)adenosine 5′-diphosphate (2Me-SADP) induced mechanical hypersensitivity and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the spinal cord. Intrathecal administration of P2Y12/P2Y13 antagonists and Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor H1152 suppressed not only p38 MAPK phosphorylation, but also mechanical hypersensitivity induced by 2Me-SADP. In the rat peripheral nerve injury model, intrathecal administration of antagonists for the P2Y12/P2Y13 receptor suppressed activation of p38 MAPK in the spinal cord. In addition, subarachnoidal injection of H1152 also attenuated nerve injury-induced spinal p38 MAPK phosphorylation and neuropathic pain behavior, suggesting an essential role of ROCK in nerve injury-induced p38 MAPK activation. We also found that the antagonists of the P2Y12/P2Y13 receptor and H1152 had inhibitory effects on the morphological changes of microglia such as retraction of processes in both 2Me-SADP and nerve injured rats. In contrast these treatments had no effect on the number of Iba1-positive cells in the nerve injury model. Collectively, our results have demonstrated roles of ROCK in the spinal microglia that is involved in p38 MAPK activation and the morphological changes. Inhibition of ROCK signaling may offer a novel target for the development of a neuropathic pain treatment.GLIA 2014
[Show abstract][Hide abstract] ABSTRACT: Xilei san (XLS), a herbal preparation widely used in China for erosive and ulcerative diseases, has been shown to be effective in ulcerative colitis (UC). The present experiments were conducted to assess its efficacy and determine its mechanism of action in a rat model that resembles human UC. The model was induced by adding 4% dextran sulfate sodium (DSS) to the rats' drinking water for 7 days. XLS was administered daily by retention enema from day 2 to day 7; the rats were sacrificed on day 8. The colon tissues were obtained for further experiments. A histological damage score and the activity of tissue myeloperoxidase were used to evaluate the severity of the colitis. The colonic cytokine levels were detected in a suspension array, and epithelial proliferation was assessed using Ki-67 immunohistochemistry. Intrarectal administration of XLS attenuated the DSS-induced colitis, as evidenced by a reduction in both the histological damage score and myeloperoxidase activity. It also decreased the levels of proinflammatory cytokines, but increased the mucosal repair-related cytokines. In addition, the epithelial Ki-67 expression was upregulated by XLS. These results suggest that XLS attenuates DSS-induced colitis by degrading proinflammatory mediators and promoting mucosal repair. XLS could be a potential topical treatment for human UC.
Evidence-based Complementary and Alternative Medicine 07/2014; 2014(3):10. DOI:10.1155/2014/569587 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear.
In the present study, activation of spinal 5-HT3 receptors by intrathecal injection of a selective 5-HT3 receptor agonist SR 57227 induced spinal glial hyperactivity, neuronal hyperexcitability and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via the chemokine fractalkine, microglia to astrocyte signaling via cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of NMDA receptors in the spinal dorsal horn. Glial hyperactivation in spinal dorsal horn after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference.
These findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neurons and glia.
[Show abstract][Hide abstract] ABSTRACT: Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knockdown of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.
Neural Regeneration Research 11/2013; 8(32):3013-9. DOI:10.3969/j.issn.1673-5374.2013.32.004 · 0.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase (COX) enzyme synthesizes prostaglandins (PGs) from arachidonic acid and exists as two major isozymes, COX-1 and COX-2. The crucial role of prostaglandins in the pathogenesis of inflammatory pain in peripheral tissue and the spinal cord has been established; however its expression dynamics after peripheral nerve injury and its role in neuropathic pain are not clear. In this study, we examined the detailed expression patterns of genes for COX, PGD2 and thromboxane A2 synthases and their receptors in the spinal cord. Furthermore, we explored the altered gene expression of these molecules using the spared nerve injury (SNI) model. We also examined whether these molecules have a role in the development or maintenance of neuropathic pain. We found a number of interesting results in this study, the first was that COX-1 was constitutively expressed in the spinal cord and up-regulated in microglia located in laminae I-II after nerve injury. Second, COX-2 mRNA expression was induced in blood vessels after nerve injury. Third, TXA2 synthase and hematopoietic PGD synthase mRNAs were dramatically increased in the microglia after nerve injury. Finally, we found that intrathecal injection of a COX-1 inhibitor and DP2 receptor antagonist significantly attenuated the mechanical allodynia. Our findings indicate that PGD2 produced by microglia is COX-1 dependent, and that neurons in the spinal cord can receive PGD2 from microglia following peripheral nerve injury. We believe that PGD2 signaling via DP2 signaling pathway from microglia to neurons is one of the triggering factors for mechanical allodynia in this neuropathic pain model.
[Show abstract][Hide abstract] ABSTRACT: Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed onset muscle soreness (DOMS), a kind of muscular mechanical hyperalgesia. Previously we reported that bradykinin-like substance released from the muscle during exercise plays a pivotal role in triggering the process of muscular mechanical hyperalgesia by upregulating nerve growth factor (NGF) in exercised muscle of rats. We show here that cyclooxygenase (COX)-2 and glial cell line-derived neurotrophic factor (GDNF) are also involved in DOMS. COX-2 inhibitors but not COX-1 inhibitors given orally before LC completely suppressed the development of DOMS, but when given 2 days after LC they failed to reverse the mechanical hyperalgesia. COX-2 mRNA and protein in exercised muscle increased 6-13 fold in mRNA and 1.7-2 fold in protein 0-12 hours after LC. COX-2 inhibitors did not suppress NGF upregulation after LC. Instead, we found GDNF mRNA was upregulated 7-8 fold in the exercised muscle 12 hours to 1 day after LC and blocked by pretreatment of COX-2 inhibitors. In situ hybridization studies revealed that both COX-2 and GDNF mRNA signals increased at the periphery of skeletal muscle cells 12 hours after LC. The accumulation of COX-2 mRNA signals was also observed in small blood vessels. Intramuscular injection of anti-GDNF antibody 2 days after LC partly reversed DOMS. Based on these findings, we conclude that GDNF upregulation through COX-2 activation is essential to mechanical hyperalgesia after exercise.
The Journal of Physiology 04/2013; 591(12). DOI:10.1113/jphysiol.2012.249235 · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Resveratrol (3,5,4' - trihydroxy-trans-stilbene), a widely distributed natural stilbenoid, was proposed to account for the unique effects of red wine on life span and health. It has been reported to possess various biological and pharmacological activities, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic effects. Here, using whole-cell patch-clamp techniques and behavioral analyses, we investigated whether resveratrol and other stilbenoids can modulate TRP channels in sensory neurons in vitro, and have analgesic effects in vivo.
We found that resveratrol dose-dependently suppressed the allyl isothiocyanate (AITC)-induced currents (IAITC) in HEK293 cells that express TRPA1, as well as in rat dorsal root ganglion (DRG) neurons. Instead, pinosylvin methyl ether (PME), another derivate of stilbene which has a similar structure to resveratrol, dose-dependently blocked the capsaicin-induced currents (ICAP) in HEK293 cells that express TRPV1 as well as in DRG neurons. Interestingly, resveratrol had no inhibitory effect on the ICAP, and PME had no effect on the IAITC. Otherwise, trans-stilbene showed no any effect on IAITC or ICAP. The concentration response curve of AITC showed that resveratrol inhibited the action of TRPA1 not by changing the EC50, but by suppressing the AITC-induced maximum response. By contrast, the inhibition of TRPV1 by PME did not change the capsaicin-induced maximum response but did cause a right shift of the EC50. Moreover, pre-administration of resveratrol suppressed intraplantar injections of AITC-evoked nocifensive behaviors, as well as that PME suppressed capsaicin-evoked one.
These data suggest that resveratrol and other stilbenoids may have an inhibitory effect on TRP channels. In addition, these stilbenoids modulate TRP channel activity in different ways.
[Show abstract][Hide abstract] ABSTRACT: Afferent fibers innervating the gastrointestinal tract have major roles in consciously evoked sensations including pain. We reported previously that the activation of ERK1/2, a member of the mitogen-activated protein kinase (MAPK) family, in primary sensory neurons was involved in acute visceral pain. Moreover, we also revealed that this activation of ERK1/2 occurred through transient receptor potential (TRP) A1, a member of the TRP family of ion channels. In contrast, it is known that the activation of p38 MAPK (p38) contributes to the development and maintenance of inflammatory and neuropathic pain. On the basis of these results, the aim of this study was to investigate the involvement of p38 and TRPA1 in acute visceral pain. Male Sprague-Dawley rats were used. Electromyographic responses to gastric distension (GD) were recorded from the acromiotrapezius muscle. We then examined the phosphorylated-p38 (p-p38) labeling in the dorsal root ganglion (DRG) after GD using immunohistochemistry. Noxious GD induced p-p38 in DRG neurons with a peak at 2 min after GD. We also found a stimulus intensity-dependent increase in the number of p-p38-immunoreactive neurons in the DRG. Intrathecal administration of the p38 inhibitor, SB203580, attenuated the electromyographic response to noxious GD. Furthermore, intrathecal administration of TRPA1 antisense oligodeoxynucleotide decreased the p38 activation in DRG neurons. The activation of p38 pathways in DRG neurons by noxious GD may be correlated with the activation state of the primary afferent neurons through TRPA1, and further, involved in the development of visceral pain.
[Show abstract][Hide abstract] ABSTRACT: Extracellular purine nucleotides and nucleosides play important roles in the nervous system, e.g., neurotransmission, neuromodulation, chemoattraction and acute inflammation. Extracellular nucleotides act through ATP receptors (P2 receptors). P2 receptors are classified into two families: the P2X receptors are ionotropic ligand-gated ion channels and the P2Y receptors are metabotropic G-protein-coupled receptors. Currently, seven P2X receptors (P2X1-7) and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) are recognized. In the sensory nervous system, ATP is suggested to be one of first mediators of tissue damage, which activates primary afferents. Nerve injury often leads to neuropathic pain, such as mechanical allodynia and painful responses to normally innocuous stimuli. Peripheral nerve injury induces the upregulation of molecules in activated microglia in the spinal cord. Microglia in the spinal cord may play an important role in the development and maintenance of neuropathic pain. A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. This review focuses on the expression of P2X and P2Y receptors mRNAs in the pain transmission pathway, i.e., in the dorsal root ganglion (DRG) and spinal cord. Furthermore, we suggest that the multiple microglial P2Y receptors activated by peripheral nerve injury may play a key role in the development of neuropathic pain.
[Show abstract][Hide abstract] ABSTRACT: Abstract Neuropathic pain syndromes are clinically characterized by spontaneous pain and evoked pain (hyperalgesia and allodynia). The optimal treatment approach for neuropathic pain is still under development because of the complex pathological mechanisms underlying this type of pain. The spinal cord is an important gateway thorough which peripheral pain signals are transmitted to the brain, and sensitization of the spinal neurons is one of the important mechanisms underlying neuropathic pain. Central sensitization represents enhancement of the function of neuronal circuits in nociceptive pathways and is a manifestation of the remarkable plasticity of the somatosensory nervous system after nerve injury. This review highlights the pathological features of central sensitization, which develops because of (1) injury-induced abnormal inputs from primary afferents, (2) increase in the excitability of dorsal horn neurons, and (3) activated glial cell-derived signals.
Brain and nerve = Shinkei kenkyū no shinpo 11/2012; 64(11):1255-65.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-18 (IL-18) is an important regulator of innate and immune responses, and is known to be expressed in various types of cells and upregulated in pathological conditions including tissue injury and inflammation, suggesting it has both proinflammatory and compensatory roles. Here we show that IL-18 was increased in microglia in the trigeminal spinal subnucleus caudalis (Vc) after peripheral nerve injury. We used a trigeminal neuropathic pain model in which the withdrawal threshold of maxillary whisker pad skin was significantly decreased after inferior alveolar nerve transection, and observed a striking increase in IL-18 expression in the Vc around the obex area from 3 d and continued until 14 d after nerve injury. The IL-18 labeled cells were largely colocalized with Iba1, suggesting this upregulation occurred in hyperactive microglia. We also found that the IL-18 induction coexisted with phosphorylated p38 MAPK, indicating a possible role of p38 in the regulation of IL-18. Our findings are the first report that injury of trigeminal nerve induced IL-18 upregulation in activated microglia in the Vc, suggesting a possible role of IL-18 in orofacial neuropathic pain.
[Show abstract][Hide abstract] ABSTRACT: A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Among the P2Y metabotropic receptors, we reported before that the P2Y12 receptor is upregulated in microglia following nerve injury and involved in the phosphorylation of p38 MAPK, and in the development of pain behavior. In this study, we examined the expression of P2Y6, P2Y13, and P2Y14 receptors in the spinal cord and whether these receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury. We found that spared nerve injury induced a dramatic increase of not only P2Y12, but also P2Y6, 13, and 14 receptor mRNA expression in spinal microglia. The increase continued for at least 2 weeks after injury. To determine whether p38 MAPK can induce the expression of P2Y receptors, we administered intrathecally the p38 MAPK inhibitor SB203580 and found that it significantly suppressed P2Y6, P2Y13, and P2Y14 but not P2Y12 mRNAs. Intrathecal injection of the specific P2Y6 antagonist MRS2578, specific P2Y13 antagonist MRS2211 or P2Y14 antisense LNA, attenuated mechanical pain hypersensitivity. The mixture of three antagonists for P2Y6, 12, and 13 showed a longer suppressive effect on pain behavior than the individual treatments. Our data demonstrate that ATP and other nucleotides may stimulate activated microglia with the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors following nerve injury and these receptors are involved in the development of neuropathic pain.