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Huntington’s disease (HD) is a progressive, neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. To investigate the metabolic alterations that occur in HD, here we examined plasma and whole‐brain metabolomic profiles of the R6/2 mouse model of HD. Plasma and brain metabolomic analyses were conducted using capillary electrophoresis–mass spectrometry (CE‐MS). In addition, liquid chromatography–mass spectrometry (LC‐MS) was also applied to plasma metabolomic analyses, to cover the broad range of metabolites with various physical and chemical properties. Various metabolic alterations were identified in R6/2 mice. We report for the first time the perturbation of histidine metabolism in the brain of R6/2 mice, which was signaled by decreases in neuroprotective dipeptides and histamine metabolites, indicative of neurodegeneration and an altered histaminergic system. Other differential metabolites were related to arginine metabolism and cysteine and methionine metabolism, suggesting upregulation of the urea cycle, perturbation of energy homeostasis, and an increase in oxidative stress. In addition, remarkable changes in specific lipid classes are indicative of dysregulation of lipid metabolism. These findings provide a deeper insight into the metabolic alterations that occur in HD and provide a foundation for the future development of HD therapeutics., Multiplatform metabolomic analysis revealed several perturbed metabolic pathways in the R6/2 mouse model of Huntington’s disease. Altered histidine metabolism in the brain indicates neurodegenerative status and an altered histaminergic system. Changes in the metabolism of arginine, cysteine, and methionine, and various lipid species were also highlighted. These findings add deeper insights into the metabolic alterations in Huntington’s disease.

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 3d and continued until 14d 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.

The L5 spinal nerve ligation (SNL) is a widely used animal neuropathic pain model. There are conflicting reports regarding the extent of injury to the L4 dorsal root ganglion (DRG) neurons in this model. If a significant number of these neurons were injured, the previously reported phenotypic and electrophysiological changes at this level are in need of re-evaluation by separating the injured neurons and the frankly spared ones. So, we immunostained activating transcription factor 3 (ATF3) and examined the change in expression of transcripts for neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF) and several voltage-gated sodium channel α-subunits (Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9) in the L4 DRG by comparing signal intensities of individual neurons using in situ hybridization histochemistry. ATF3-immunoreactivity was similarly observed in 4-6% of neuronal nuclei of the SNL and sham-operated ipsilateral L4 DRGs. Comparison between ATF3+ and ATF3- neurons in the SNL L4 DRG revealed that (1) whereas NPY induction occurred in ATF3+ cells, BDNF increased mainly in ATF3- neurons; (2) although ATF3+ neurons had higher Nav1.3 signals than ATF3- neurons, these signals were much lower than those of the L5 DRG neurons; and (3) ATF3+/N52- neurons selectively lost Nav1.8 and Nav1.9 mRNAs. Comparison of the total neuronal populations among naïve, SNL, and sham-operated rats revealed no significant differences for all examined Nav mRNAs. Because neuropathic pain behaviors were developed by rats with SNL but not the sham-operation, the small number of injured L4 neurons likely do not contribute to the pathomechanisms of neuropathic pain.

Recent studies indicate that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3receptor 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-HT3receptor and its contribution to facilitation of pain remain unclear. In the present study, activation of spinal 5-HT3receptor by intrathecal injection of a selective 5-HT3receptor agonist, SR57227, induced spinal glial hyperactivity, neuronal hyperexcitability, and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via chemokine fractalkine, microglia to astrocyte signaling via the cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of GluN (NMDA) receptors in the spinal dorsal horn. In addition, exogenous brain-derived neurotrophic factor-induced descending pain facilitation was accompanied by upregulation of CD11b and GFAP expression in the spinal dorsal horn after microinjection in the RVM, and these events were significantly prevented by functional blockade of spinal 5-HT3receptors. Enhanced expression of spinal CD11b and GFAP after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Thus, 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 neuron and glia.

Insomnia is a common problem for people with chronic pain. Cortical GABAergic neurons are part of the neurobiological substrate that underlies homeostatic sleep regulation. In the present study, we confirmed that sciatic nerve ligation caused thermal hyperalgesia and tactile allodynia in mice. In this experimental model for neuropathic pain, we found an increase in wakefulness and a decrease in non-rapid eye movement sleep under a neuropathic pain-like state. Under these conditions, membrane-bound GABA (γ-aminobutyric acid) transporters (GATs) on activated glial fibrillary acidic protein-positive astrocytes were significantly increased in the cingulate cortex, and extracellular GABA levels in this area after depolarization were rapidly decreased by nerve injury. Furthermore, sleep disturbance induced by sciatic nerve ligation was improved by the intracingulate cortex injection of a GAT-3 inhibitor. These findings provide novel evidence that sciatic nerve ligation decreases extracellular-released GABA in the cingulate cortex of mice. These phenomena may, at least in part, explain the insomnia in patients with neuropathic pain. Neuropathic pain-like stimuli suppress the GABAergic transmission with increased GABA (γ-aminobutyric acid) transporters located on activated astrocytes in the cingulate cortex related to sleep disturbance.

A modification of the message site in the skeleton of naltrexone was carried out to improve the potency and selectivity of the compound for an opioid receptor subtype. In the course of conversion, we synthesized 7-membered ring ether derivatives, which had an inserted OCH(2) group between 4- and 6-positions of morphinan skeleton. One of the 7-membered ring ether derivatives possessed more potent antagonistic activity than naltrexone for the mu opioid receptor. Another compound possessing 17-methyl group derived from noroxycodone may be a mu opioid receptor partial agonist and showed analgesic activity. We are currently examining the subtype selectivity of these compounds.

Background & Aims: Changes in the properties of visceral sensory neurons contribute to the development of gastrointestinal pain. However, little is known about the molecules involved in mechanosensation from the gastrointestinal tract. We investigated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase cascade, in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons by noxious gastric distention (GD) and its involvement in acute visceral pain in rats. Methods: Electromyographic responses to gastric balloon distention through gastrostomy were recorded from the acromiotrapezius muscle in rats after splanchnic nerve resection or vagotomy and in control rats. We then examined the phosphorylated-ERK1/2 (p-ERK1/2) labeling in the DRG and NG after GD using immunohistochemistry. Results: Gastric distention induced p-ERK1/2 in DRG and NG neurons with a peak at 2 minutes after stimulation. We found a stimulus intensity-dependent increase in the number of activated neurons, and this activation corresponded well with the incidence of the visceromotor response. Most of these p-ERK1/2–labeled neurons were small- and medium-sized neurons that coexpressed transient receptor potential vanilloid 1 ion channel and acid-sensing ion channel 3. Splanchnic nerve resection, but not vagotomy, affected the visceromotor response, and attenuated the ERK1/2 activation in DRG neurons produced by GD. Furthermore, intrathecal administration of the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126, altered the response to noxious GD. Conclusions: The activation of ERK1/2 pathways in DRG neurons by noxious GD may be correlated with functional activity, and may be involved in acute visceral pain.

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

Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain. © 2008 Wiley-Liss, Inc.

The present study was undertaken to investigate pharmacological actions induced by morphine and oxycodone under a neuropathic pain-like state. In the mu-opioid receptor (MOR) binding study and G-protein activation, we confirmed that both morphine and oxycodone showed MOR agonistic activities. Mice with sciatic nerve ligation exhibited the marked neuropathic pain-like behavior. Under these conditions, antinociception induced by subcutaneously (s.c.) injected morphine was significantly decreased by sciatic nerve ligation, whereas s.c. injection of oxycodone produced a profound antinociception in sciatic nerve-ligated mice. There were no significant differences in spinal or supraspinal antinociception of morphine and oxycodone between sham operation and nerve ligation. Moreover, either morphine- or oxycodone-induced increase in guanosine-5'-o-(3-thio) triphosphate ([(35)S]GTPgammaS) binding in the spinal cord, periaqueductal gray matter and thalamus in sciatic nerve-ligated mice was similar to that in sham-operated mice. Antinociception induced by s.c., intrathecal, or intracerebroventricular injection of the morphine metabolite morphine-6-glucuronide (M-6-G) was significantly decreased by sciatic nerve ligation. Furthermore, the increase in the G-protein activation induced by M-6-G was eliminated in sciatic nerve ligation. In addition, either morphine- or oxycodone-induced rewarding effect was dramatically suppressed under a neuropathic pain-like state. The increased [(35)S]GTPgammaS binding by morphine or oxycodone was significantly lower in the lower midbrain of mice with sciatic nerve ligation compared with that in control mice. These findings provide further evidence that oxycodone shows a profound antinociceptive effect under a neuropathic pain-like state with less of a rewarding effect. Furthermore, the reduction in G-protein activation induced by M-6-G may, at least in part, contribute to the suppression of the antinociceptive effect produced by morphine under a neuropathic pain-like state.

The central mechanisms of neuropathic pain following chronic ethanol consumption are poorly understood. We previously reported that the levels of metabotropic glutamate 5 (mGlu5) receptor and phosphorylated-protein kinase C (PKC) were significantly increased in the spinal cord following chronic ethanol consumption. The aim of this study was to investigate whether mGlu5 receptor and PKC inhibitors directly attenuate the neuropathic pain-like state induced by chronic ethanol treatment in rats. A significant decrease in the mechanical nociceptive threshold was observed 5 weeks of chronic ethanol consumption. This hyperalgesia was significantly attenuated by repeated i.p. injection of (S)-2,6-diamino-N-[[1-(oxotridecyl)-2-piperidinyl]methyl] hexanamide dihydrochloride (NPC15437), a selective PKC inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, this hyperalgesia was also significantly attenuated by repeated i.p. injection of 6-methyl-2-[phenylethynyl]-pyridine (MPEP), a selective mGlu5 receptor inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, the hyperalgesia that developed after 5 weeks of ethanol treatment was significantly suppressed by a single i.p. post-injection with either NPC15437 or MPEP. These findings constitute direct evidence that spinal mGlu5 receptor and PKC play substantial roles in the development and maintenance of an ethanol-dependent neuropathic pain-like state in rats.

The present study was undertaken to investigate the role of spinal voltage-dependent calcium channel alpha(2)delta-1 subunit in the expression of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. In cultured spinal neurons, gabapentin (GBP), which displays the inhibitory effect of alpha(2)delta-1 subunit, suppressed the extracellular Ca(2+) influx induced by KCl, whereas it failed to inhibit the intracellular Ca(2+) release induced by inositol-1,4,5-triphosphate. Seven days after sciatic nerve ligation, the protein level of alpha(2)delta-1 subunit in the ipsilateral spinal cord was clearly increased compared to that observed in sham-operated mice. In addition, the mRNA level of alpha(2)delta-1 subunit was significantly increased in the dorsal root ganglion, but not in the spinal cord, of nerve-ligated mice. Under these conditions, a marked decrease in the latency of paw-withdrawal against a thermal stimulation and tactile stimulation, induced by sciatic nerve ligation was abolished by repeated intrathecal (i.t.) treatment with GBP. Additionally, the persistent reduction in the nociceptive threshold by i.t. treatment with GBP at the early stage of the neuropathic pain-like state was maintained for 7 days even after GBP withdrawal. It is of interest to note that a single i.t. post-injection of GBP showed a marked and transient inhibitory effect on the developed neuropathic pain-like state, whereas repeated i.t. post-treatment with GBP produced a persistent inhibitory effect during the treatment. In conclusion, we propose here that the neuropathic pain-like state with sciatic nerve ligation is associated with the increased level of the alpha(2)delta-1 subunit of Ca(2+) channels at the sensory nerve terminal in the spinal dorsal horn of mice. Furthermore, the present data provide evidence that the neuropathic pain may be effectively controlled by repeated treatment with GBP at the early stage.

Chronic ethanol consumption produces painful neuropathy for which there is no reliably successful therapy, largely due to a lack of understanding of the central mechanisms that underlie the development of the neuropathic pain-like state induced by chronic ethanol treatment. The aim of this study was to investigate what mechanisms contribute to the neuropathic pain-like state induced by chronic ethanol treatment in rats. Mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal, and lasted for 14 weeks. This hyperalgesia was significantly attenuated by repeated i.p. injection of ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist. Under these conditions, mRNA and protein levels of NR1, NR2A and NR2B subunits did not change in the spinal cord of chronic ethanol-fed rats. Interestingly, phosphorylated-Ser-1303 NR2B (p-Ser1303-NR2B) subunit was significantly increased in the spinal cord of chronic ethanol-fed rats, whereas p-Tyr1472-NR2B was not affected in the superficial spinal dorsal horn of ethanol-fed rats. These findings suggest that spinal p-Ser1303-NR2B plays a significant role in the development of the ethanol-dependent neuropathic pain-like state in rats.

Clinically, it has been reported that chronic pain induces depression, anxiety, and reduced quality of life. The endogenous opioid system has been implicated in nociception, anxiety, and stress. The present study was undertaken to investigate whether chronic pain could induce anxiogenic effects and changes in the opioidergic function in the amygdala in mice. We found that either injection of complete Freund's adjuvant (CFA) or neuropathic pain induced by sciatic nerve ligation produced a significant anxiogenic effect at 4 weeks after the injection or surgery. Under these conditions, the selective mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin (DAMGO)- and the selective delta-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)-stimulated [35S]GTPgammaS binding in membranes of the amygdala was significantly suppressed by CFA injection or nerve ligation. CFA injection was associated with a significant increase in the kappa-opioid receptor agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]acetamide hydrochloride (ICI199,441)-stimulated [35S]GTPgammaS binding in membranes of the amygdala. The intracerebroventricular administration and microinjection of a selective mu-opioid receptor antagonist, a selective delta-opioid receptor antagonist, and the endogenous kappa-opioid receptor ligand dynorphin A caused a significant anxiogenic effect in mice. We also found that thermal hyperalgesia induced by sciatic nerve ligation was reversed at 8 weeks after surgery. In the light-dark test, the time spent in the lit compartment was not changed at 8 weeks after surgery. Collectively, the present data constitute the first evidence that chronic pain has an anxiogenic effect in mice. This phenomenon may be associated with changes in opioidergic function in the amygdala.

Tetrodotoxin-sensitive (TTX-s) spontaneous activity is recorded from the dorsal roots after peripheral nerve injury. Primary sensory neurons in the dorsal root ganglion (DRG) express multiple TTX-s voltage-gated sodium channel α-subunits (Navs). Since Nav1.3 increases, whereas all other Navs decrease, in the DRG neurons after peripheral nerve lesion, Nav1.3 is proposed to be critical for the generation of these spontaneous discharges and the contributions of other Navs have been ignored. Here, we re-evaluate the changes in expression of three other TTX-s Navs, Nav1.1, Nav1.6 and Nav1.7, in the injured 5th lumbar (L5) primary afferent components following L5 spinal nerve ligation (SNL) using in situ hybridization histochemistry and immunohistochemistry. While the overall signal intensities for these Nav mRNAs decreased, many injured DRG neurons still expressed these transcripts at clearly detectable levels. All these Nav proteins accumulated at the proximal stump of the ligated L5 spinal nerve. The immunostaining patterns of Nav1.6 and Nav1.7 associated with the nodes of Ranvier were maintained in the ipsilateral L5 dorsal root. Interestingly, putative proprioceptive neurons characterized by α3 Na+/K+ ATPase-immunostaining specifically lacked Nav1.7 mRNA in naive DRG but displayed de novo expression of this transcript following SNL. Nav1.7-immunoreactive fibers were significantly increased in the ipsilateral gracile nucleus where central axonal branches of the injured A-fiber afferents terminated. These data indicate that multiple TTX-s channel subunits could contribute to the generation and propagation of the spontaneous discharges in the injured primary afferents. Specifically, Nav1.7 may cause some functional changes in sensory processing in the gracile nucleus after peripheral nerve injury.

Background: Transient receptor potential (TRP)A1, a member of the TRP family of ion channels, has been proposed to function in diverse sensory processes, including thermosensation and pain. However, TRPA1 has not been directly implicated in stomach mechanosensation, and its contribution to acute visceral pain from this organ is unknown. Here, we investigated the expression of TRPA1 in primary sensory afferents and its involvement in visceral hypersensitivity in rats. Methods: We examined TRPA1 expression in the dorsal root ganglion (DRG), nodose ganglion (NG), and stomach of rats by using immunohistochemistry. Electromyographic responses to gastric distention (GD) were recorded from the acromiotrapezius muscle in TRPA1 knockdown rats and in control rats. Results: TRPA1 was predominantly expressed with sensory neuropeptides in DRG and NG neurons, and in nerve fibres in the rat stomach. Gastric distention induced the activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in DRG and NG neurons 2 min after stimulation, and most of the phosphorylated-ERK1/2-labelled DRG neurons were TRPA1-positive neurons. Intrathecal injection of TRPA1 antisense attenuated the visceromotor response, and suppressed ERK1/2 activation in the DRG, but not NG, neurons produced by GD. Furthermore, intrathecal and intraperitoneal injections of the TRPA1 inhibitor HC-03003 suppressed the response to noxious GD. Conclusions: The activation of TRPA1 in DRG neurons by noxious GD may be involved in acute visceral pain. Our findings point to the potential blockade of TRPA1 in primary afferents as a new therapeutic target for the reduction of visceral hypersensitivity.

In this paper, the optimization of the suspension was performed by using the criterion taking account of human sensitivity in order to improve the riding comfort. For the purpose of decreasing the vibration at an uncomfortable frequency range for human beings, the frequency-shaped criterion was formulated. An active suspension with feedback and feedforward controls was designed by using the optimal control theory from the criterion. The suspension model is assumed to be a two-degree-of freedom system, and the acceleration of road unevenness is taken to be colored noise. From numerical calculations and experiments, it was made clear that optimal control with a feedback link alone obtained from the frequency-shaped criterion function can reduce the acceleration of a vehicle at the desired frequency range. Furthermore, it was clarified that the control with a feedforward link is more useful than the one with a feedback link alone in the same control force.

Interleukin (IL)-18 is an important regulator of innate and acquired immune responses. Here we show that both the IL-18 and IL-18 receptor (IL-18R), which are induced in spinal dorsal horn, are crucial for tactile allodynia after nerve injury. Nerve injury induced a striking increase in IL-18 and IL-18R expression in the dorsal horn, and IL-18 and IL-18R were upregulated in hyperactive microglia and astrocytes, respectively. The functional inhibition of IL-18 signaling pathways suppressed injury-induced tactile allodynia and decreased the phosphorylation of nuclear factor κB in spinal astrocytes and the induction of astroglial markers. Conversely, intrathecal injection of IL-18 induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Our results indicate that IL-18-mediated microglia/astrocyte interactions in the spinal cord have a substantial role in the generation of tactile allodynia. Thus, blocking IL-18 signaling in glial cells might provide a fruitful strategy for treating neuropathic pain.

A novel 6,14-epoxymorphinan benzamide derivative (NS22) was synthesized, which showed opioid kappa receptor agonistic activity in the [(35)S]GTPgammaS binding assay. The antinociceptive effect of NS22 was evaluated in the tail-flick and the hot-plate test. This compound showed a potent antinociceptive activity in mice by s.c. administration, which was attenuated with nor-BNI (selective opioid kappa receptor antagonist).

The aim of the present study was to investigate the role of protein kinases within the spinal cord in the development of a neuropathic pain-like state induced by partial sciatic nerve ligation in mice. Thermal hyperalgesia induced by nerve ligation in mice was markedly suppressed by either repeated intrathecal (i.t.) pre-treatment or post-treatment with the selective protein kinase (PKC) inhibitor RO-32-0432 and the selective Rho kinase inhibitor Y-27632. In contrast, sciatic nerve ligation-induced thermal hyperalgesia was not observed by repeated i.t. pre-treatment with the selective PKA inhibitor KT5720. Interestingly, thermal hyperalgesia induced by nerve ligation in mice was significantly suppressed by repeated i.t. post-treatment with fasudil, which possesses the inhibitory effect of several protein kinases including PKC and Rho kinase. Collectively, these findings suggest that a long-lasting activation of PKC and RhoA/Rho kinase pathways in the spinal cord may be responsible for the development of thermal hyperalgesia induced by nerve ligation in mice. The present data raise the fascinating possibility that i.t. or epidural administration with fasudil may be useful for the treatment of neuropathic pain.

The present study was designed to investigate the rewarding effects induced by tramadol and its active metabolite O-desmethyltramadol (M1) under a neuropathic pain-like state.In opioid receptor binding and G protein activation, we confirmed that M1, but not tramadol, showed mu-opioid receptor (MOR) agonistic activity. Furthermore, we found that the subcutaneous (s.c.) injection of tramadol and M1 each produced a significant place preference in mice, and these effects were significantly suppressed by pretreatment with the MOR antagonist beta-funaltrexamine. The dopamine level in the mouse nucleus accumbens was significantly increased by s.c. injection of either tramadol or M1. Mice with sciatic nerve ligation exhibited a marked decrease in the latency of paw withdrawal in response to a thermal stimulus only on the ipsilateral side. Under these neuropathic pain-like conditions, the rewarding effect induced by s.c. injection of either tramadol or M1 was dramatically inhibited after sciatic nerve ligation. Furthermore, the M1-induced G protein activation in the lower midbrain area was suppressed after sciatic nerve ligation.Our present data support the notion that the rewarding effect induced by tramadol is mediated mainly through metabolism to its active metabolite M1 via MOR. Furthermore, the suppression of the M1-induced G protein activation in the lower midbrain area caused by sciatic nerve ligation may be responsible for inhibiting the rewarding effects induced by s.c. injection of tramadol and M1 under a neuropathic pain-like state.

The present study was undertaken to investigate the possible change in anti-hyperalgesic effect following repeated treatment with morphine or fentanyl using the dose to improve the thermal hyperalgesia under an inflammatory pain-like state. The anti-hyperalgesic effect induced by fentanyl in complete Freund's adjuvant (CFA)-pretreated mice rapidly disappeared during the consecutive administration of fentanyl, whereas morphine preserved its potency of anti-hyperalgesic effect. In addition, repeated treatment with fentanyl, but not morphine, resulted in the increase in levels of phosphorylated-mciro-opioid receptor (MOR) associated with the enhanced inactivation of protein phosphatase 2A and the reduction in Rab4-dependent MOR resensitization. Next, we investigated the specific involvement of the opioid receptor types and MOR subtypes in analgesic properties of morphine and fentanyl in the mouse spinal cord. In the competitive displacement binding assay with [1H]DAMGO, no significant difference in the binding affinity to MOR between morphine and fentanyl was noted in membranes obtained from the mouse spinal cord. Furthermore, there was no significant difference between morphine and fentanyl in either antinociceptive effect or G-protein activation in mice partially lacking MOR-1B, which shows a greater resistance to agonist-induced desensitization than for other MOR subtypes. These findings point out the possibility that the chronic treatment with fentanyl may cause the different modulation from chronic treatment with morphine on either the internalization or resensitization of MORs in the spinal cord under a pain-like state. The present data provide the first evidence for the mechanism underlying the development of tolerance to fentanyl-induced anti-hyperalgesic effect under chronic pain.

Central mechanisms of neuropathy induced by chronic ethanol treatment are almost unknown. In this study, rats were treated with ethanol-diet for 72 days. Mechanical hyperalgesia was observed during ethanol consumption, even after ethanol withdrawal. Under these conditions, a microglial marker ionized calcium-binding adaptor molecule 1-, but not a neuron marker microtuble associated protein-2-, like immunoreactivies were increased in the rat spinal cord. Furthermore, hypertrophy of microglia was clearly observed following chronic ethanol consumption. These findings support the idea that the activation and hypertrophy of microglia in the spinal cord may be, at least in part, associated with in the induction of ethanol-dependent neuropathic pain-like state.

The present study was undertaken to further clarify the role of tyrosine phosphorylation of NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptor in the development of the morphine-induced rewarding effect in mice. The morphine (5 mg/kg, sc)-induced rewarding effect was completely inhibited by pretreatment with a selective NR2B subunit-containing NMDA receptor antagonist ifenprodil (20 mg/kg, i.p.). The protein level of phospho-Tyr-1472, but not phospho-Ser-1303, NR2B subunit was significantly increased in the mouse limbic forebrain containing the nucleus accumbens (N.Acc.) of mice that had shown the morphine-induced rewarding effect. In addition, the level of phospho-Tyr-416 Src family kinase was also increased in the limbic forebrain of mice that had shown the morphine-induced rewarding effect. These findings suggest that Tyr-1472 phosphorylation of NR2B subunit-containing NMDA receptor associated with activation of Src family kinase in the limbic forebrain may be involved in the morphine-induced rewarding effect.

Alcohol neuropathy has been thought to involve decreased nerve function following chronic ethanol consumption. However, there is no reliably successful therapy, largely due to a lack of understanding of the central underlying mechanisms. The aim of this study was to investigate the mechanisms that contribute to the neuropathic pain-like state induced by chronic ethanol treatment in rats. Rats were chronically treated with ethanol diet (1.25-5% of ethanol) for over 70 days. Mechanical hyperalgesia was observed during ethanol consumption and even after ethanol withdrawal. Under these conditions, an immunohistochemical study showed an increase in metabotropic glutamate receptor 5 (mGluR5) immunoreactivity in the superficial spinal dorsal horn of chronic ethanol-fed rats. Furthermore, immunoblot analysis revealed that the protein level of mGluR5 was clearly increased following chronic ethanol consumption. These findings support the idea that the increased levels of mGluR5 in the spinal cord may be, at least in part, involved in the induction of ethanol-dependent neuropathic pain-like state.

A growing body of evidence indicates that the mesolimbic dopaminergic (DAergic) pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.) play a critical role in the initiation of psychological dependence on morphine. As well as DAergic system, the involvement of non-DAergic neurotransmitter and neuromodulator systems in rewarding effects induced by morphine has been recently documented. We previously demonstrated that the morphine-induced rewarding effect was dramatically suppressed by co-treatment with NMDA receptor antagonists, such as dizocilpine (MK-801), ketamine and ifenprodil. Therefore, we propose here that inhibiting the N-methyl-D-aspartate (NMDA) receptor and its associated protein kinase in the N.Acc. is useful for the treatment for psychological dependence on morphine. The following review provides a summary of recent our findings regarding the role of NMDA receptor and its associated protein kinase in the development of psychological dependence on morphine.

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. Results 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. Conclusions 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.