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Bupivacaine (Bup) has a certain research basis in pain-related diseases, but it has not been studied in painful diabetic neuropathy. In this study, we investigated the role of Bupivacaine in painful diabetic neuropathy. Mouse model with painful diabetic neuropathy was established, and then treated with different concentrations of Bupivacaine. The blood glucose level in the tail vein and the changes in body weight was measured. The mechanical allodynia, thermal hyperalgesia and thermal allodynia was assessed by pain behavioral tests. Microglia were treated with high glucose (HG) and different concentrations of Bupivacaine. The levels of inflammatory cytokines were detected by using Enzyme-linked immunosorbent assays. Dual luciferase reporter assay explored the relationship between miR-23a and phosphodiesterase 4 B (PDE4B). The results displayed that Bupivacaine ameliorated the mechanical allodynia, thermal hyperalgesia, and thermal allodynia in mice with painful diabetic neuropathy, and is more effective at low concentration. Moreover, low concentration of Bupivacaine inhibited inflammation and promoted miR-23a expression in mice with painful diabetic neuropathy and in microglia induced by HIGH GLUCOSE. Overexpression of miR-23a reduced the levels of inflammatory cytokines by down-regulating PDE4B expression. Knockdown of miR-23a reversed the inhibition effect of Bupivacaine on microglial inflammation. These results revealed that low concentration of Bupivacaine inhibited microglial inflammation through down-regulating PDE4B via miR-23a, thereby attenuated painful diabetic neuropathy., (Copyright © 2020. Published by Elsevier B.V.)

Background: Neuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury., Methods: A total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1β-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1β, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay., Results: We found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3'UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1., Conclusions: JHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis., (Copyright © 2020. Published by Elsevier B.V.)

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults. While it is primarily characterized by the death of upper and lower motor neurons, there is a significant metabolic component involved in the progression of the disease. Two-thirds of ALS patients have metabolic alterations that are associated with the severity of symptoms. In ALS, as in other neurodegenerative diseases, the metabolism of glycosphingolipids, a class of complex lipids, is strongly dysregulated. We therefore assume that this pathway constitutes an interesting avenue for therapeutic approaches. We have shown that the glucosylceramide degrading enzyme, glucocerebrosidase (GBA) 2 is abnormally increased in the spinal cord of the SOD1 G86R mouse model of ALS. Ambroxol, a chaperone molecule that inhibits GBA2, has been shown to have beneficial effects by slowing the development of the disease in SOD1 G86R mice. Currently used in clinical trials for Parkinson's and Gaucher disease, ambroxol could be considered as a promising therapeutic treatment for ALS., (Copyright © 2020. Published by Elsevier B.V.)

We have previously reported that the spinal angiotensin (Ang) system is involved in the modulation of streptozotocin (STZ)-induced diabetic neuropathic pain in mice. An important drawback of this model however is the fact that the neuropathic pain is independent of hyperglycemia and produced by the direct stimulation of peripheral nerves. Here, using the leptin deficient ob/ob mouse as a type 2 diabetic model, we examined whether the spinal Ang system was involved in naturally occuring diabetic neuropathic pain. Blood glucose levels were increased in ob/ob mice at 5-15 weeks of age. Following the hyperglycemia, persistent tactile and thermal hyperalgesia were observed at 11-14 and 9-15 weeks of age, respectively, which was ameliorated by insulin treatment. At 12 weeks of age, the expression of Ang-converting enzyme (ACE) 2 in the spinal plasma membrane fraction was decreased in ob/ob mice. Spinal ACE2 was expressed in neurons and microglia but the number of NeuN-positive neurons was decreased in ob/ob mice. In addition, the intrathecal administration of Ang (1-7) and SB203580, a p38 MAPK inhibitor, attenuated hyperalgesia in ob/ob mice. The phosphorylation of spinal p38 MAPK was also attenuated by Ang (1-7) in ob/ob mice. These inhibitory effects of Ang (1-7) were prevented by A779, a Mas receptor antagonist. In conclusion, we revealed that the Ang (1-7)-generating system is downregulated in ob/ob mice and is accompanied by a loss of ACE2-positive neurons. Furthermore, Ang (1-7) decreased the diabetic neuropathic pain through inhibition of p38 MAPK phosphorylation via spinal Mas receptors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The protein kinase C family regulates many cellular functions, including multiple forms of neuroplasticity. The novel PKCθ and atypical PKCζ isoforms have been implicated in distinct forms of spinal, respiratory motor plasticity, including phrenic motor facilitation (pMF) following acute intermittent hypoxia or inactivity, respectively. Although these PKC isoforms are critical in regulating spinal motor plasticity, other isoforms may be important for phrenic motor plasticity. We tested the impact of conventional/novel PKC activator, phorbol 12-myristate 13-acetate (PMA) on pMF. Rats given cervical intrathecal injections of PMA exhibited pMF, which was abolished by pretreatment of broad-spectrum PKC inhibitors bisindolymalemide 1 (BIS) or NPC-15437 (NPC). Because PMA fails to activate atypical PKC isoforms, and NPC does not block PKCθ, this finding demonstrates that classical/novel PKC isoforms besides PKCθ are sufficient to elicit pMF. These results advance our understanding of mechanisms producing respiratory motor plasticity, and may inspire new treatments for disorders that compromise breathing, such as ALS, spinal injury and obstructive sleep apnea., (Copyright © 2017. Published by Elsevier B.V.)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving motor neurons in the motor cortex, brainstem and spinal cord. ALS leads to progressive, aggravated muscle weakness and paralysis. Although the precise pathogenesis remains unknown, several studies have shown that estrogens exert neuroprotective effects during the course of the disease. Aromatase is the key enzyme in estrogen synthesis. In the present study, we used immunohistochemistry, immunofluorescence and western blotting to observe the characteristics of aromatase expression in the spinal cords of copper-zinc superoxide dismutase-1 (SOD1)-G93A transgenic mice. Under normal and nearly normal (pre-symptomatic stage) conditions, the motor neurons in the spinal anterior horn expressed aromatase. After disease onset, astrocytes began to express aromatase. The total level of aromatase expression decreased with disease progression. These findings may provide the basis for the pathogenesis of ALS through glial aromatization during the progression of this disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis (MS), inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Clinically manifested EAE can be induced in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats by immunization with spinal cord homogenate (SCH) and complete Freund's adjuvant (CFA). Matrix metalloproteinases (MMP) play important roles in various steps of MS and EAE pathogenesis. Expression of gelatinases MMP2 and MMP9, their activator MMP14 and their inhibitor tissue inhibitor of MMP (TIMP)1 in the CNS of AO and DA rats immunized with SCH+CFA was determined. Expression of mRNA for MMP2, MMP9 and MMP14 was higher and expression of TIMP1 mRNA was lower in AO rats. However, gelatinase activity in spinal cords was higher in samples obtained from DA rats. Further, while there was no strain difference in MMP2 and MMP9 mRNA expression in lymph nodes of the immunized rats, gelatinase activity was higher in DA rats. This activity was reduced by antiinflammatory cytokines interleukin (IL)-10 and IL-4. Interestingly, gelatinase activity was detected in the nuclei of cells within the CNS, but not of those in lymph nodes. Our results imply that posttranscriptional regulation of MMP2 and MMP9 expression and/or function determines low gelatinase activity within the CNS and in immune cells of EAE-resistant AO rats., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freund's adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EA's analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia., Results: We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn., Conclusions: Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EA's antinociception in the inflammatory pain model., (Copyright © 2014 Elsevier Inc. All rights reserved.)

N-methyl-D-aspartate receptor (NMDAR) and metabotropic glutamate receptor 5 (mGluR5) play an important role in nociceptive processing and central sensitization. Our previous study showed that tyrosine phosphorylation of NMDAR subunit 2B (NR2B) at Tyr1472 in spinal dorsal horn contributes to the postoperative hyperalgesia induced by remifentanil. Cyclin-dependent kinase 5 (Cdk5) has been implicated in synaptic plasticity, learning, memory and pain signaling via regulating the phosphorylation of NMDAR and mGluR5. In the present study, a rat model of postoperative pain was used to investigate the role of Cdk5 in spinal dorsal horn in remifentanil-induced hyperalgesia and the intervention of pretreatment with Cdk5 inhibitor roscovitine. Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous) significantly enhanced mechanical allodynia and thermal hyperalgesia induced by plantar incision during the postoperative period (each lasting between 2 h and 48 h), which were attenuated by pretreatment with roscovitine. Correlated with the pain behavior changes, Western blotting revealed that there was a significant increase in the expression of Cdk5 and its activator p35/p25, and further the kinase activity of Cdk5 in spinal dorsal horn after intraoperative infusion of remifentanil. The phosphorylation of NR2A at Ser1232, the phosphorylation of NR2B at Tyr1472 and the phosphorylation of mGluR5 at Ser1167 were also significantly up-regulated. Furthermore, these increases were attenuated by pretreatment with roscovitine. These results suggested that Cdk5 may contribute to remifentanil-induced postoperative hyperalgesia via regulating the phosphorylation of NMDAR and mGluR5 in spinal dorsal horn. These findings provide experimental evidence for the further application of Cdk5 inhibitor in preventing remifentanil-induced hyperalgesia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fentanyl has been widely used in anesthesia and analgesia, especially for cardiovascular surgeries. The aim of the study was to evaluate whether remote intrathecal fentanyl preconditioning (RFPC) provides cardioprotection and the role of spinal nitric oxide synthase (NOS) system in this effect. Fentanyl (0.3μg/kg) was administered intrathecally during RFPC by 3 cycles of 5-minute infusions interspersed with 5-minute infusion free periods. A non-specific nitric oxide synthase (NOS) inhibitor NG-nitro l-arginine methyl ester (l-NAME, 30nmol) and a selective nNOS inhibitor 7-nitroindazole (7-NI, 100nmol) were administered intrathecally 10min before RFPC, and were used to evaluate the involvement of the NOS system of the spinal cord. RFPC group markedly reduced the infarct size compared with control. However, the cardioprotection of RFPC could be abolished by pretreatment with l-NAME and 7-NI. RFPC merely increased the expression of nNOS and did not affect iNOS and eNOS expression. l-NAME reversed nNOS expression up-regulation induced by RFPC treatment. The present study demonstrated that RFPC effectively induced cardioprotection through activating the nNOS in the spinal cord., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phrenic long-term facilitation (pLTF) is a form of serotonin-dependent respiratory plasticity induced by acute intermittent hypoxia (AIH). pLTF requires spinal Gq protein-coupled serotonin-2 receptor (5-HT2) activation, new synthesis of brain-derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, TrkB. Intrathecal injections of selective agonists for Gs protein-coupled receptors (adenosine 2A and serotonin-7; 5-HT7) also induce long-lasting phrenic motor facilitation via TrkB "trans-activation." Since serotonin released near phrenic motor neurons may activate multiple serotonin receptor subtypes, we tested the hypothesis that 5-HT7 receptor activation contributes to AIH-induced pLTF. A selective 5-HT7 receptor antagonist (SB-269970, 5mM, 12 μl) was administered intrathecally at C4 to anesthetized, vagotomized and ventilated rats prior to AIH (3, 5-min episodes, 11% O2). Contrary to predictions, pLTF was greater in SB-269970 treated versus control rats (80 ± 11% versus 45 ± 6% 60 min post-AIH; p<0.05). Hypoglossal LTF was unaffected by spinal 5-HT7 receptor inhibition, suggesting that drug effects were localized to the spinal cord. Since 5-HT7 receptors are coupled to protein kinase A (PKA), we tested the hypothesis that PKA inhibits AIH-induced pLTF. Similar to 5-HT7 receptor inhibition, spinal PKA inhibition (KT-5720, 100 μM, 15 μl) enhanced pLTF (99 ± 15% 60 min post-AIH; p<0.05). Conversely, PKA activation (8-br-cAMP, 100 μM, 15 μl) blunted pLTF versus control rats (16 ± 5% versus 45 ± 6% 60 min post-AIH; p<0.05). These findings suggest a novel mechanism whereby spinal Gs protein-coupled 5-HT7 receptors constrain AIH-induced pLTF via PKA activity., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The analgesic effectiveness of long-term opioid therapies is compromised by the development of antinociceptive tolerance linked to the overt production of peroxynitrite (ONOO(-), PN), the product of the interaction between superoxide (O2(-), SO) and nitric oxide (NO), and to neuroinflammatory processes. We have recently reported that in addition to post-translational nitration and inactivation of mitochondrial manganese superoxide dismutase (MnSOD), activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme (NOX) in the spinal cord is a major source for the overt production of superoxide-derived PN during the development of morphine-induced antinociceptive tolerance. However, the NOX complex involved in these processes is not known. The objective of these studies is to identify a potential role for the NOX2 complex, an enzyme involved in inflammation. Mice lacking the catalytic subunit of NOX2 (Nox2(-/-)) or its regulatory subunit, p47(phox) (p47(phox)(-/-)), developed antinociceptive tolerance similar to wildtype (wt) mice after 3 days of continuous morphine. However, while wt mice continue to develop tolerance by day six, morphine analgesia was restored in both Nox2(-/-) and p47(phox)(-/-) mice. Moreover, the loss of Nox2 or p47 did not affect acute morphine analgesia in naïve mice. In wt mice, antinociceptive tolerance was associated with increased activation of NOX, nitration of MnSOD, and proinflammatory cytokines production in the spinal cord. These events were markedly attenuated in Nox2(-/-) and p47(phox)(-/-) mice and instead, there was enhanced formation of antiinflammatory cytokine (IL4 and IL10) production. These results suggest that NOX2 activity provides a significant source of superoxide-derived PN to undertake post-translational modifications of mitochondrial MnSOD and to engage neuroinflammatory signaling in the spinal cord associated with opioid-induced antinociceptive tolerance. Thus, NOX2 may provide a potential target for adjuvant therapy to protect opioid analgesia., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The cholinergic system plays important roles in neurotransmission in both the peripheral and central nervous systems. The cholinergic neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and its action terminated by acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The predominance of AChE has focused much attention on understanding the relationship of this enzyme to ChAT-positive cholinergic neurons. However, there is ample evidence that BuChE also plays an important role in cholinergic regulation. To elucidate the relationship of BuChE to neural elements that are producing acetylcholine, the distribution of this enzyme was compared to that of ChAT in the mouse CNS. Brain tissues from 129S1/SvImJ mice were stained for BuChE and ChAT using histochemical, immunohistochemical and immunofluorescent techniques. Both BuChE and ChAT were found in neural elements throughout the CNS. BuChE staining with histochemistry and immunohistochemistry produced the same distribution of labeling throughout the brain and spinal cord. Immunofluorescent double labeling demonstrated that many nuclei in the medulla oblongata, as well as regions of the spinal cord, had neurons that contained both BuChE and ChAT. BuChE-positive neurons without ChAT were found in close proximity with ChAT-positive neuropil in areas such as the thalamus and amygdala. BuChE-positive neuropil was also found closely associated with ChAT-positive neurons, particularly in tegmental nuclei of the pons. These observations provide further neuroanatomical evidence of a role for BuChE in the regulation of acetylcholine levels in the CNS., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The phosphodiesterases (PDEs) are a superfamily of enzymes that regulate spatio-temporal signaling by the intracellular second messengers cAMP and cGMP. PDE2A is expressed at high levels in the mammalian brain. To advance our understanding of the role of this enzyme in regulation of neuronal signaling, we here describe the distribution of PDE2A in the rat brain. PDE2A mRNA was prominently expressed in glutamatergic pyramidal cells in cortex, and in pyramidal and dentate granule cells in the hippocampus. Protein concentrated in the axons and nerve terminals of these neurons; staining was markedly weaker in the cell bodies and proximal dendrites. In addition, in both hippocampus and cortex, small populations of non-pyramidal cells, presumed to be interneurons, were strongly immunoreactive. PDE2A mRNA was expressed in medium spiny neurons in neostriatum. Little immunoreactivity was observed in cell bodies, whereas dense immunoreactivity was found in the axon tracts of these neurons and their terminal regions in globus pallidus and substantia nigra pars reticulata. Immunostaining was dense in the medial habenula, but weak in other diencephalic regions. In midbrain and hindbrain, immunostaining was restricted to discrete regions of the neuropil or clusters of cell bodies. These results suggest that PDE2A may modulate cortical, hippocampal and striatal networks at several levels. Preferential distribution of PDE2A into axons and terminals of the principal neurons suggests roles in regulation of axonal excitability or transmitter release. The enzyme is also in forebrain interneurons, and in mid- and hindbrain neurons that may modulate forebrain networks and circuits., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

There is ample evidence of the biological changes produced by the sustained activation of opioid receptors. We evaluated the adaptive changes of cerebral Na(+),K(+)-ATPase in response to the sustained administration of morphine (minipumps, 45mg/kg/day, 6 days) in CD-1 mice and the functional role of these changes in opioid antinociception. The antinociceptive effect of morphine as determined with tail-flick tests was reduced in morphine-tolerant mice. There were no significant changes in the density of high-affinity Na(+),K(+)-ATPase α subunits labeled with [(3)H]ouabain in forebrain membranes from morphine-tolerant compared to those of morphine-naive animals. Western blot analysis showed that there were no significant differences between groups in the changes in relative abundance of α(1) and α(3) subunits of Na(+),K(+)-ATPase in the spinal cord or forebrain. However, the morphine-induced stimulation of Na(+),K(+)-ATPase activity was significantly lower in brain synaptosomes from morphine-tolerant mice (EC(50)=1.79±0.10μM) than in synaptosomes from morphine-naive mice (EC(50)=0.69±0.12μM). Furthermore, adaptive alterations in the time-course of basal Na(+),K(+)-ATPase activity were observed after sustained morphine treatment, with a change from a bi-exponential decay model (morphine-naive mice) to a mono-exponential model (morphine-tolerant mice). In behavioral studies the antinociceptive effects of morphine (s.c.) in the tail-flick test were dose-dependently antagonized by ouabain (1 and 10ng/mouse, i.c.v.) in morphine-naive mice, but not in morphine-tolerant mice. These findings suggest that during morphine tolerance, adaptive cellular changes take place in cerebral Na(+),K(+)-ATPase activity which are of functional relevance for morphine-induced antinociception., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spinal metabotropic glutamate receptor 5 (mGlu₅ receptor) is known to influence the development of intrathecal morphine antinociceptive tolerance. However, the signaling mechanisms remain unknown. We carried out intrathecal administration of an antisense oligodeoxynucleotide (ODN), which results in reduced expression of spinal mGlu₅ receptor, to determine its effects on morphine tolerance and spinal protein kinase C (PKC) expression. Rats were treated intrathecally with saline, morphine, mGlu5 receptor antisense ODN or mGlu5 receptor mismatched ODN. Behavioral tests were used to test the thermal and mechanical pain thresholds. Eight days later, rats were sacrificed and spinal cords were harvested to assess the expression of spinal PKC (α, γ and ε) by Western blotting and real-time polymerase chain reaction (PCR). Compared to control, intrathecal mGlu₅ receptor antisense ODN resulted in a ~53.9% reduction of spinal mGlu₅ receptor after 8days treatment. The mGlu5 receptor antisense ODN prevented the development of morphine tolerance. Expression of spinal PKC (α, γ and ε) was up-regulated at the mRNA and protein levels during the development of tolerance. Meanwhile, antisense ODN but not mismatched ODN reduced the spinal dorsal horn levels of PKC (α, γ and ε) which had been up-regulated after morphine exposure. We conclude that mGlu₅ receptor participates in the development of morphine tolerance. Expression of spinal PKC (α, γ and ε) at the mRNA and protein levels increased during morphine tolerance. Antisense ODN of mGlu₅ receptor prevented the tolerance and inhibited the altered expression of spinal PKC (α, γ and ε) during the development of tolerance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca²⁺, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative-nitrosative stress, an important trigger of mitogen activated protein kinase (MAPK) phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16 mg kg⁻¹ d⁻¹, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S)-hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-Lipoxygenase gene deficiency prevented diabetes-associated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase⁻/⁻ mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fatty acid amide hydrolase (FAAH) is an enzyme that metabolizes endocannabinoids and fatty acid amides possibly linked to activation of the opioid system. To examine how this enzyme affects spinal signalling, electrophysiological recordings in the dorsal horn and qPCR on dorsal horn tissue following systemic administration of the FAAH inhibitor URB597 (0.3 and 1.0mg/kg i.v.) and spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.), were performed. The present data showed that the suppressive effect of the FAAH inhibitor URB597 (1.0mg/kg i.v.) on the spinal nociceptive responses was prevented by spinal administration of the opioid receptor antagonist naloxone (0.1 μg/μl i.th.). Moreover, the present findings demonstrated that the FAAH inhibitor URB597 (1.0mg/kg i.v.) partly reversed expression of spinal long-term potentiation (LTP) and also attenuated the LTP-associated increased Zif expression. We conclude that pharmacological inactivation of FAAH may be a promising strategy to inhibit the development of central hyperalgesia; thereby reinforcing the role of FAAH as a potential therapeutic target., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study investigated the antinociceptive effect of intrathecal escin and examined its effect on the formalin-induced activation of c-Fos and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the rat spinal cord. Rats were chronically implanted with lumbar intrathecal catheters, and the ability of intrathecal escin to alter nociceptive behaviours in the rat formalin test was examined. The expression of c-Fos and p-p38 MAPK in the dorsal horn of the spinal cord was detected in the control and escin (40μg) groups using immunohistochemical techniques. Intrathecal escin produced a dose-dependent reduction in formalin-evoked flinching behaviour in rats during the second phase; however, no effect was observed in the first phase. In addition, immunohistochemical experiments showed that the expression of c-Fos and p-p38 MAPK in the spinal cord dorsal horn increased after an injection of formalin into the paw. Interestingly, the 40μg dose of intrathecal escin, which was the larger of the two doses that blocked formalin-induced hyperalgaesia, attenuated the formalin-induced increases in c-Fos and p-p38 MAPK in the dorsal horn of the spinal cord. The decrease in pain-related behaviours and c-Fos expression indicated that escin produced antinociceptive effects in the rat formalin test. Although the specific mechanisms of these effects were not investigated, the reduction in p-p38 MAPK in the dorsal horn of the spinal cord may be involved., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) have been implicated in the modulation of synaptic plasticity, glial activation, and long-term potentiation in the CNS. Here we demonstrate for the first time a mechanism for the regulation of nociceptive processing by spinal MMP-3 during peripheral inflammation. We first determined by western blotting that the catalytic (active) form of MMP-3 (cMMP-3) is increased in lumbar spinal cord following peripheral inflammation in rats. The peripheral inflammation-induced thermal hyperalgesia and tactile hypersensitivity was transiently (2-3 h) attenuated by intrathecal (IT) pretreatment with either an MMP-3 inhibitor (NNGH), or a broad spectrum MMP inhibitor (GM6001). In addition, IT delivery of cMMP-3 evoked hypersensitivity, whereas the pro (enzymatically inactive) form of MMP-3 did not. This suggests a pro-algesic effect of spinal MMP-3 mediated by an enzymatic mechanism. This cMMP-3-induced hypersensitivity is concurrent with increased tumor necrosis factor (TNF) in the spinal cord. The hypersensitivity behavior is prevented by intrathecal etanercept (TNF blockade). Treatment with cMMP-3 resulted in an increase in TNF release from spinal primary microglial, but not astrocyte cultures. These findings thus present direct evidence implicating MMP-3 in the coordination of spinal nociceptive processing via a spinal TNF-dependent mechanism., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinases (MAPKs) are important signaling factors in many cellular processes including cell proliferation and survival during development and synaptic plasticity induced by acute nociception in the adult. There is extensive evidence for the involvement of members of the MAPK family, the extracellular signal-regulated kinases 1 and 2 (ERKs 1/2), in the development of acute inflammatory somatic and visceral pain, but their role in the maintenance of chronic pain states is unknown. We have previously shown that ovariectomy of adult mice (OVX) generates a persistent and estrogen-dependent abdominal hyperalgesic state that lasts for several months and is not related to a persistent nociceptive afferent input. Here we have used OVX mice to study a possible role of ERK 1/2 in the spinal processing of this form of chronic abdominal hyperalgesia. Eight weeks after OVX the mice showed a robust abdominal hyperalgesia and a significant increase in the activation of ERK1/2 in the lumbosacral spinal cord. This enhanced activation was not seen in control and sham-operated mice or in regions of the cord other than lumbosacral in OVX mice. Also, the increased activation of ERK 1/2 observed in OVX mice matched the time course of the hyperalgesic state as no activation was observed at week 1 after OVX when the hyperalgesic state had not yet developed. Administration of slow-release pellets containing 17β-estradiol at week 5 post OVX reversed both the development of the hyperalgesia and the enhanced activation of ERK 1/2, suggesting that this activation, like the hyperalgesic state, was estrogen-dependent. Intrathecal injections of the ERK 1/2 inhibitor U0126 successfully rescued the mice from the abdominal hyperalgesia for up to 24 h after the injection and also reversed the enhanced expression of ERK 1/2. Our study shows, for the first time, activation of ERK 1/2 in the spinal cord matching the time course of an estrogen-dependent chronic hyperalgesic state., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The neonate opioid system has been frequently investigated, and studies have shown that exposure to drugs in early life can have implications for nervous system development. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in central and peripheral mechanisms of nociception. Extracellular nucleotides can be hydrolyzed by E-NTPDases and ecto-5'nucleotidase, which present the functions of removing ATP and generating adenosine. In this study, we evaluated ATP, ADP, and AMP hydrolysis in synaptosomes from spinal cord and cerebral cortex of rats at postnatal day 16 after repeated morphine exposure in early life (postnatal day 8 to 14). Additionally, we evaluated E-NTPDase (1, 2 and 3) and ecto-5'nucleotidase gene expression by semi-quantitative RT-PCR analysis. We observed an increase in ATP hydrolysis in the cerebral cortex, and a decrease in ADP hydrolysis in spinal cord. Expression levels of E-NTPDase 1 decreased in cerebral cortex and increased in spinal cord. Our findings highlight the importance of the purinergic system in young rats submitted to repeated morphine exposure by showing that in the neonatal period such exposure is capable of affecting the control system for nucleotide levels, which can promote changes in modulation or transmission of painful stimuli., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The gamma isoform of protein kinase C (PKCgamma) is an injury-activated intracellular modulator that boosts neuronal activity in algesic and neuroregenerative signalling pathways. Acetyl-L-carnitine (ALCAR), a physiological compound with role in bioenergetic functions, shows an antihyperalgesic effect and at the same time can exert neuroregenerative and neuroprotective effects. Aimed to explore the link between pain and neuroregeneration, the effect of ALCAR treatment (100 mg kg(-1) i.p. twice daily for 15 days) on PKCgamma and mitogen-activated protein kinases (MAPKs) expression has been evaluated in CCI (chronic constriction injury) rats. The sciatic nerve and the lumbar tract of the spinal cord were processed to evaluate the levels of the phosphorylated form of PKCgamma, ERK 1,2, SAP/JNK, p-38 and c-Jun; furthermore, the mRNA expression of the early genes c-Jun and c-Fos has been investigated. Fifteen days after injury, the analysis in the sciatic nerves highlighted a bilateral increase of the activated forms of PKCgamma, ERK 1,2 and SAP/JNK, whereas c-Jun showed an increase only ipsilaterally. ALCAR completely prevented mechanical hyperalgesia and provoked in the nerve a c-Jun increment only. In the lumbar tract of the spinal cord, higher levels of activated PKCgamma, ERK 1,2, p38, SAP/JNK and c-Jun proteins were detected in the ipsilateral side in respect of sham. ALCAR was able to stimulate this expression profile. At the transcriptional level c-Jun mRNA was increased in the ipsilateral side of spinal cord of CCI saline-treated rats, whereas c-Fos mRNA was unchanged. ALCAR had a stimulatory effect on both these early genes. These findings may represent a different approach in the study of the complex balance between pain and neuroregeneration and could constitute the basis for developing new disease modifying agents in the treatment of neuropathic pain.

Previous studies have implicated nitric oxide (NO) in the antinociceptive response to the anesthetic gas nitrous oxide (N(2)O). The present study was conducted to confirm this NO involvement using pharmacological and gene knockdown and knockout strategies to inhibit the supraspinal and spinal production of NO. Antinociceptive responsiveness to 70% N(2)O was assessed using the acetic acid (0.6%) abdominal constriction test in NIH Swiss mice following intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with the NOS-inhibitor l-N(G)-nitro arginine methyl ester (L-NAME) or an antisense oligodeoxynucleotide (AS-ODN) directed against neuronal NOS (nNOS). Experiments were also conducted in mice homozygous for a defective nNOS gene (nNOS(-/-)). Mice that were pretreated i.c.v. or i.t. with L-NAME (1.0 microg) both exhibited 80-90% reduction in the magnitude of the N(2)O-induced antinociceptive response. Mice that were pretreated i.c.v. or i.t. with nNOS AS-ODN (3 x 25microg) exhibited a 60-80% antagonism of the antinociceptive response. Compared to wild-type mice, nNOS knockout mice showed a 60% reduction in N(2)O-induced antinociception. These findings consistently demonstrate that transient or developmental suppression of nNOS expression significantly reduces antinociceptive responsiveness to N(2)O. NO of both supraspinal and spinal origin, therefore, plays an important role in the antinociceptive response to N(2)O.

Glutathione (GSH) is a major non-enzymatic antioxidant which is present in all tissues. Its protective actions occur through different pathways such its role as a substrate of antioxidant enzymes, such as glutathione peroxidase (GPx) and glutathione-S-transferase (GST). Nitric oxide (NO) is involved in many physiological processes in the central nervous system, including nociception. In spite of much evidence concerning oxidative and nitrosative stress and neuropathic pain, the exact role of these molecules in pain processing is still unknown. Sciatic nerve transection (SNT) was employed to induce neuropathic pain in rats. Glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities, glutathione (GSH) content, GSH/GSSG ratio, nitric oxide metabolites (NOx) and neuronal nitric oxide synthase (nNOS) protein expression in the lumbosacral spinal cord were determined. All of these analyses were performed in the SNT and sham groups 1, 3, 7 and 15 days after surgery. There was an increase in GPx activity and in GSH content 3 days after surgery in both sham and SNT groups, but the GSH/GSSG ratio increased only in the SNT group in this time point. nNOS expression was upregulated 7 days post SNT. NOx was detected 1 day after surgery in sham and SNT groups, but at 7 and 15 days, the increase occurred only in SNT animals. These results support the role of the gluthatione system in pain physiology and highlight the involvement of NO as an important molecule related to nociception.

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine. Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region are transcribed from the cholinergic gene locus from different promoter regions and produced by alternative splicing in the mouse. Digoxigenin-labeled riboprobes and in situ hybridization histochemistry were used to investigate the expression of N1, R1, R2, R3, R4 and total choline acetyltransferase mRNA in the mouse CNS. The relative levels of choline acetyltransferase transcripts differed dramatically in distinct subdivisions of the mature cholinergic nervous system. Neurons hybridizing with antisense riboprobes for all of the five investigated splice variants (R1, R2, R3, R4 and N1) as well as those hybridizing with riboprobe for the common protein-coding region were found in a number of expected regions in the CNS. They include the basal forebrain, striatum, pontomesencephalic tegmentum, motor and autonomic nuclei of the brainstem, and spinal cord. Neurons with a moderate to very high level of expression of R1 and R2 splice variants were distributed in both the forebrain and brainstem nuclei. On the other hand, R3, R4 and N1 splice variants revealed a moderate to high level of expression in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord compared to a low expression level in forebrain cholinergic structures. No expression of the N1, R1, R2, R3 and R4 splice variants was detectable in the neurons of the cerebral cortex, hippocampus and medial habenular nucleus. With the riboprobe for the common protein-coding region, the neurons of the medial habenular nucleus could be labeled at high level, while intrinsic cortical neurons were labeled at low level. Hippocampus revealed no significant hybridization for total choline acetyltransferase mRNA. These findings strongly suggested that: (1) R1 and R2 were the major splice variants expressed in the neurons of forebrain nuclei; (2) R1, R2, R3, R4 and N1 splice variants were almost equally expressed in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord; (3) inferring from a paucity of other isoforms, M type choline acetyltransferase mRNA is a splice variant predominantly expressed in the cerebral cortex and medial habenular nucleus.

Electroacupuncture (EA) can effectively control the exaggerated pain in humans with inflammatory disease and animals with experimental inflammatory pain. However, there have been few investigations on the effect of co-administration of EA and analgesics and the underlying synergistic mechanism. Using behavioral test, RT-PCR analysis, enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA), the present study demonstrated that (1) Unilateral intra-articular injection of complete Freund's adjuvant (CFA) produced a constant hyperalgesia and an up-regulation of the prostaglandin E(2) (PGE(2)) level as well as the tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 levels in the spinal cord; (2) Celecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2), at a dose of 2, 10, and 20 mg/kg (twice daily, p.o.), presented a dose-dependent anti-hyperalgesic effect; (3) Repeated EA stimulation of ipsilateral 'Huan-Tiao' (GB30) and 'Yang-Ling-Quan' (GB34) acupoints significantly suppressed CFA-induced hyperalgesia, and markedly inhibited the CFA-induced increase of the level of PGE(2) as well as IL-1beta, IL-6, and TNF-alpha in the spinal cord; (4) EA combined with low dose of celecoxib (2 mg/kg, twice daily, p.o.) greatly enhanced the anti-hyperalgesic effects of EA, with a synergistic reversing effect on CFA-induced up-regulation of spinal PGE(2), but not on the IL-1beta, IL-6, or TNF-alpha. These data indicated that repeated EA combined with low dose of celecoxib produced synergistic anti-hyperalgesic effect in the CFA-induced monoarthritic rats, which could be made possible by regulating the activity of spinal COX, hence the spinal PGE(2) level. Thus, this combination may provide an effective strategy for pain management.

This study investigated the effect of 5th and 6th lumbar nerve (L5/L6) spinal nerve ligation (SNL) on activated nuclear factor kappaB (NFkBa) in nuclear extracts from the lumbar dorsal horn of the rat, and its relationship to prostaglandin (PG)-dependent spinal hyperexcitability and allodynia 3 days later. Male Sprague-Dawley rats, fitted with intrathecal (i.t.) catheters, underwent SNL- or sham-surgery. Paw withdrawal threshold (PWT), electromyographic analysis of the biceps femoris flexor reflex, and immunoblotting of the spinal cord were used. Both allodynia (PWT

Electroacupuncture (EA) has long been used to treat pain including neuropathic pain, but its mechanisms remain to be delineated. Since cyclooxygenase-2 (COX-2) has been reported to increase in the spinal dorsal horn following spinal nerve ligation (SNL) and it may play a role in the neuropathic pain, we hereby tested the hypothesis that EA may affect COX-2 expression and hence neuropathic nociception after SNL. The results showed that EA (2 Hz) can significantly reduce mechanical and thermal hypersensitivity following lumbar L5 SNL in rats. Immunostaining demonstrated suppression of COX-2 expression in the spinal L4-L6 dorsal horn after EA. The present results suggest that EA may alleviate neuropathic hypersensitivity by, at least partially, inhibiting COX-2 expression in the spinal cord.

Activation of the spinal phospholipase A(2) (PLA(2)) -cyclooxygenase (COX) -prostaglandin signaling pathway is widely implicated in nociceptive processing. Although the role of spinal COX isoforms in pain signal transmission has been extensively characterized, our knowledge of PLA(2) enzymes in this cascade is limited. Among all PLA(2) groups, cytosolic calcium-dependent PLA(2) group IVA (cPLA(2)IVA) appears to be the predominant PLA(2) enzyme in the spinal cord. In the present study we sought to (i) characterize anatomical and cellular distribution and localization of cPLA(2)IVA in dorsal horn of rat spinal cord, (ii) verify efficacy and selectivity of intrathecal (IT) delivery of an antisense oligonucleotide (AS) targeting rat cPLA(2)IVA mRNA on spinal expression of this enzyme, and (iii) examine the effect of down-regulation of spinal cPLA(2)IVA on peripheral tissue injury-induced pain behavior. Here we demonstrate that cPLA(2)IVA is constitutively expressed in rat spinal cord, predominantly in dorsal horn neurons and oligodendrocytes but not in astrocytes or microglia. Intrathecal injection of AS significantly down-regulated both protein and gene expression of cPLA(2)IVA in rat spinal cord, while control missense oligonucleotide (MS) had no effect. Immunocytochemistry confirmed that the reduction occurred in neurons and oligodendrocytes. cPLA(2)IVA AS did not alter expression of several other PLA(2) isoforms, such as secretory PLA(2) (groups IIA and V) and calcium-independent PLA(2) (group VI), indicating that the AS was specific for cPLA(2)IVA. This selective knockdown of spinal cPLA(2)IVA did not change acute nociception (i.e. paw withdrawal thresholds to acute thermal stimuli and intradermal formalin-induced first phase flinching), however, it significantly attenuated formalin-induced hyperalgesia (i.e. second phase flinching behavior), which reflects spinal sensitization. Thus the present findings suggest that cPLA(2)IVA may specifically participate in spinal nociceptive processing.

Various animal models of pain are dependent on activation of different glutamate receptor subtypes. First degree burn of the paw elicits a secondary hyperalgesia that is dependent on Ca2+ permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not N-methyl-D-aspartate (NMDA) receptors. The present study takes advantage of that specificity by examining the effects of spinal pretreatments of agents on this secondary hyperalgesia. Rats with indwelling intrathecal catheters were pretreated with agents prior to paw injury. Mechanical withdrawal thresholds were measured before, and for three h after the injury. Spinal pretreatment with cyclooxygenase (10 and 30 microg (S)-(+)-ibuprofen; and 3 and 30 microg ketorolac) and nitric oxide synthase (33 and 100 microg N(G) Nitro-L-arginine methyl ester hydrochloride (L-NAME) and 10 microg thiocitrulline) inhibitors resulted in no specific anti-allodynia. In contrast, ziconotide (0.3, 1.0 and 3 microg), the N-type voltage gated calcium channel antagonist was very effective in blocking burn-induced sensitivity at all doses used. l-type (Diltiazam 230 microg) and P-type (Agatoxin IVA 0.3 microg) calcium channel blockers produced intermediate effects. Thus, cyclooxygenase and nitric oxide synthase are assumed not to be downstream of Ca2+ permeable AMPA receptors. Voltage gated calcium channels blockers could exert their effects either pre- or post-synaptically.

Protein kinase C gamma (PKCgamma) is widely distributed throughout the CNS and is thought to play a role in long term hyper-excitability in nociceptive neurones. Here, we provide the first report of PKCgamma cells in the dorsal column nuclei of the adult rat. Retrograde labeling of PKCgamma cells from the thalamus with choleragenoid revealed that 25% of the PKCgamma positive gracile cells projected to the thalamus. Further, we have characterized the distribution of PKCgamma within gracile nucleus in terms of colocalization with various neurotransmitter receptors or enzymes and calcium binding proteins, and compared this with PKCgamma colocalization in cells of laminae I-III of the spinal cord. We show that approximately 90% of the PKCgamma cells in the gracile nucleus and 60% in the dorsal horn were immuno-positive for the AMPA receptor subunit glutamate 2/3 (GluR2/3). Little coexpression was seen with neurokinin 1 receptor, nitric oxide synthase (NOS) and the AMPA receptor subunit GluR1, markers of distinct neuronal subpopulations. In the spinal cord, a quarter of PKCgamma cells expressed calbindin, but very few cells did so in the gracile nucleus. Electrical stimulation at c-fiber strength of the normal or injured sciatic nerve was used to induce c-fos as a marker of postsynaptic activation in the spinal cord and gracile nucleus. Quantitative analysis of the number of PKCgamma positive gracile cells that expressed also c-fos increased from none to 24% after injury, indicating an alteration in the sensory activation pattern in these neurones after injury. C-fos was not induced in inner lamina II following c-fiber electrical stimulation of the intact or axotomized sciatic nerve, indicating no such plasticity at the spinal cord level. As dorsal column nuclei cells may contribute to allodynia after peripheral nerve injury, pharmacological modulation of PKCgamma activity may therefore be a possible way to ameliorate neuropathic pain after peripheral nerve injury.

Granulocyte colony-stimulating factor (G-CSF) is a potent hematopoietic factor. Recently, this factor has been shown to exhibit neuroprotective effects on many CNS injuries. Spinal cord ischemic injury that frequently results in paraplegia is a major cause of morbidity after thoracic aorta operations. In the present study, we examined the neuroprotective role of G-CSF on spinal cord ischemia-induced neurological dysfunctions and changes in the mitogen-activated protein kinase (MAPK) and Akt signaling pathways in the spinal cord. Spinal cord ischemia was induced in male Wistar rats by occluding the descending aorta with a 2F Fogarty catheter for 12 min 30 s. Immediately after ischemia surgery, the rats were administered G-CSF (10 mug) or saline by intrathecal (i.t.) injection. The rats were divided into four groups: control, ischemia plus saline, ischemia plus G-CSF and G-CSF alone. The neurological dysfunctions were assessed by calculating the motor deficit index after ischemia surgery. The expressions of MAPK and Akt were studied using Western blotting and double immunohistochemistry. First, we observed that ischemia plus i.t. G-CSF can significantly reduce the motor function defects and downregulate phospho-p38 and phospho-c-Jun N-terminal kinase protein expressions-this can be compared with the ischemia plus saline group. In addition, G-CSF inhibited the ischemia-induced activation of p38 in the astrocytes. Furthermore, we concluded that i.t. G-CSF produced a significant increase in phospho-Akt and phospho-ERK in the motor neurons and exhibited beneficial effects on the spinal cord ischemia-induced neurological defects.

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2 pathway may play an important role in the development, but not maintenance, of chronic pain following peripheral tissue inflammation.

In spite of prominent progress in basic pain research, neuropathic pain remains a significant medical problem, because it is often poorly relieved by conventional analgesics. Thus this situation encourages us to make more sophisticated efforts toward the discovery of new analgesics. We previously showed that i.t. administration of acromelic acid-A (ACRO-A), a Japanese mushroom poison, provoked prominent tactile pain (allodynia) at an extremely low dose of 1 fg/mouse. In the present study we synthesized ACRO-A analogues (2S,3R,4R)-3-carboxymethyl-4-phenoxypyrrolidine-2-carboxylic acid (POPA-2) and (2S,3R,4R)-3-carboxymethyl-4-(phenylthio)pyrrolidine-2-carboxylic acid (PSPA-1) chemically and examined their ability to induce allodynia in conscious mice. Whereas POPA-2 induced allodynia at extremely low doses from 1 to 100 fg/mouse, similar to ACRO-A, PSPA-1 did not induce allodynia; rather, it inhibited the ACRO-A-induced allodynia with an ID(50) value (95% confidence limits) of 2.19 fg/mouse (0.04-31.8 fg/mouse). Furthermore, PSPA-1 relieved neuropathic pain produced by L5 spinal nerve transection on day 7 after the operation in a dose-dependent manner from 1 to 100 pg/mouse. In contrast, it did not affect thermal or mechanical nociception or inflammatory pain. PSPA-1 reduced the increase in neuronal nitric oxide synthase activity in the spinal cord of neuropathic pain mice assessed by NADPH-diaphorase histochemistry and blocked the allodynia induced by N-methyl-d-aspartate. These results demonstrate that PSPA-1 may represent a novel class of anti-allodynic agents for neuropathic pain acting by blocking the glutamate-nitric oxide pathway.

Mutations in Cu,Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). It has been proposed that neuronal cell death might occur due to inappropriately increased Cu interaction with mutant SOD1. Using Cu immobilized metal-affinity chromatography (IMAC), we showed that mutant SOD1 (A4V, G85R, and G93A) expressed in transfected COS7 cells, transgenic mouse spinal cord tissue, and transformed yeast possessed higher affinity for Cu than wild-type SOD1. Serine substitution for cysteine at the Cys111 residue in mutant SOD1 abolished the Cu interaction on IMAC. C111S substitution reversed the accelerated degradation of mutant SOD1 in transfected cells, suggesting that the Cys111 residue is critical for the stability of mutant SOD1. Aberrant Cu binding at the Cys111 residue may be a significant factor in altering mutant SOD1 behavior and may explain the benefit of controlling Cu access to mutant SOD1 in models of familial ALS.

Dnmt3a and Dnmt3b, which are known as functional de novo methyltransferases, are responsible for creating genomic methylation patterns during mammalian development. Recently, we have shown that specific expression of Dnmt3b in epiblast, embryonic ectoderm, hematopoietic progenitor cells and spermatogonia cells is followed by Dnmt3a expression (Watanabe D, Suetake I, Tada T, Tajima S (2002) Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev 118:187-190; Watanabe D, Suetake I, Tajima S, Hanaoka K (2004) Expression of Dnmt3b in mouse hematopoietic progenitor cells and spermatogonia at specific stages. Gene Expr Patterns 5:43-49). In this study, we analyzed the expression of mouse de novo methyltransferases during development of the nervous systems. In the embryonic olfactory epithelium (OE), Dnmt3b was specifically expressed in Mash1 positive globose basal cells (i.e. transiently amplifying neural progenitor cells), while Dnmt3a was expressed in immature olfactory receptor neurons. Dnmt3b-positive cells were rarely observed in the adult OE, but were increased in regenerating OE with intranasal ZnSO(4) administration. Dnmt3b was also detected in the E8.5 neural plate, E10.5 spinal cord and retina cells, while Dnmt3a was expressed in postmitotic young neurons. Furthermore, Dnmt3b was specifically expressed in ES cells, while Dnmt3a was transiently expressed during neural cell differentiation of ES cells. Dnmt3b is specifically expressed in progenitor cells during hematopoiesis, spermatogenesis and neurogenesis, suggesting an important role in the initial steps of progenitor cell differentiation. Dnmt3a is expressed in postmitotic young neurons following the Dnmt3b expression. Dnmt3a may be required for the establishment of tissue-specific methylation patterns of the genome. The coordinated expression of de novo methyltransferases from Dnmt3b to Dnmt3a suggests conserved mechanisms of de novo methylation of the genome and different functions for Dnmt3b and Dnmt3a during progenitor cell development.

The possible antinociceptive effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), was investigated in mice by using the hot-plate and abdominal constriction response (writhing) tests. In addition, the expression of Rho-kinase protein (ROCK-2) was studied in the mouse brain and spinal cord by Western blotting. Male balb/c mice (n=8, for each group) were used in the experiment. Hot-plate latency and the number of writhes were recorded in control and in Y-27632-treated (1-5 mg/kg, i.p.) groups. Y-27632 (1 mg/kg) did not affect hot-plate latency; however, it considerably diminished the number of writhes, from 89+/-12 in control to 30+/-6 in the mice treated with 1 mg/kg Y-27632 (P=0.001). At a higher dose (5 mg/kg), Y-27632 prolonged the hot-plate latency from 8.7+/-1.0 s to 14.4+/-1.7 s (P=0.005) and decreased the number of writhes from 80+/-8 to 24+/-7 (P=0.002). Western blot analysis revealed that mouse spinal cord and brain homogenates expressed ROCK-2 protein. These results indicate that Rho-kinase may be involved in nociception and that its inhibitors, such as Y-27632, may represent a new type of antinociceptive drug.

Mutations in reeler, the gene coding for the Reelin protein, result in pronounced motor deficits associated with positioning errors (i.e. ectopic locations) in the cerebral and cerebellar cortices. In this study we provide the first evidence that the reeler mutant also has profound sensory defects. We focused on the dorsal horn of the spinal cord, which receives inputs from small diameter primary afferents and processes information about noxious, painful stimulation. We used immunocytochemistry to map the distribution of Reelin and Disabled-1 (the protein product of the reeler gene, and the intracellular adaptor protein, Dab1, involved in its signaling pathway) in adjacent regions of the developing dorsal horn, from early to late embryonic development. As high levels of Dab1 accumulate in cells that sustain positioning errors in reeler mutants, our findings of increased Dab1 immunoreactivity in reeler laminae I-III, lamina V and the lateral spinal nucleus suggest that there are incorrectly located neurons in the reeler dorsal horn. Subsequently, we identified an aberrant neuronal compaction in reeler lamina I and a reduction of neurons in the lateral spinal nucleus throughout the spinal cord. Additionally, we detected neurokinin-1 receptors expressed by Dab1-labeled neurons in reeler laminae I-III and the lateral spinal nucleus. Consistent with these anatomical abnormalities having functional consequences, we found a significant reduction in mechanical sensitivity and a pronounced thermal hyperalgesia (increased pain sensitivity) in reeler compared with control mice. As the nociceptors in control and reeler dorsal root ganglia are similar, our results indicate that Reelin signaling is an essential contributor to the normal development of central circuits that underlie nociceptive processing and pain.

Hyperalgesia following unilateral complete Freund's adjuvant-induced inflammation was characterized by paw withdrawal latency to thermal stimulus. Paw withdrawal latencies were significantly shorter on the complete Freund's adjuvant-treated paw than on the contralateral paw of the complete Freund's adjuvant- and the sham-treated rats. Total cytosolic protein kinase C activity in the lumbar enlargement was unchanged on the sides of the spinal cord ipsi- and contra-lateral to the inflamed paw. Membrane-associated activities of protein kinase Calpha, protein kinase CbetaI and protein kinase Cgamma did not change significantly on the sides of the cord ipsi- and contra-lateral to the inflammation. However, membrane-associated activity of protein kinase CbetaII was increased in the cord section ipsilateral to the inflammation, suggesting that increased translocation/activation of protein kinase CbetaII is related to thermal hyperalgesia. Dextrorphan (an N-methyl-D-aspartate receptor antagonist), L-703,606 (an NK-1 receptor antagonist) and an antisense oligodeoxynucleotide for a selective knockdown of protein kinase Cbeta, reduced complete Freund's adjuvant-induced hyperalgesia, and reversed significant changes in the membrane activity of protein kinase CbetaII on the spinal cord section ipsilateral to the inflamed paw. Dextrorphan and protein kinase Cbeta antisense oligodeoxynucleotide were effective in reversing complete Freund's adjuvant-induced increase in the activity of protein kinase CbetaII ipsilateral to the inflammation at all the doses tested, but L-703,606 was effective only at the highest dose. Furthermore, in the presence of inflammatory stimulus, dextrorphan and L-703,606 did not alter the activities of membrane-associated protein kinase Calpha, protein kinase CbetaI, and protein kinase Cgamma in the section of the spinal cord ipsi- and contra-lateral to the inflammation. Protein kinase Cbeta antisense oligodeoxynucleotide had no significant effect on the membrane-associated activities of protein kinase Calpha and protein kinase Cgamma, but decreased the activities of both protein kinase CbetaI and protein kinase CbetaII and the expression of protein kinase Cbeta isozyme in the spinal cord. The data provide evidence that a common molecular event that converges to initiate and maintain hyperalgesia may include the translocation and activation of protein kinase CbetaII in the spinal dorsal horn.

The present study investigated the effect of different levels of Delta-9-tetrahydrocannabinol (Delta(9)-THC) antinociceptive tolerance on Protein Kinase A (PKA) activity in mouse brain and spinal cord. To strengthen this investigation, a positive control was developed to demonstrate the assay utilized in this study was sensitive enough to detect an increase in PKA activity in the anatomical regions utilized in this study. The membrane-permeant and phosphodiesterase-resistant cAMP analog 8-Bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8-Br-cAMPS) was utilized for the development of this positive control and this compound produced an increase in PKA activity in several mouse brain regions (i.c.v.) and lumbar spinal cord (i.t.) following its administration. Models were then developed in which mice expressed either a 13-fold or 49-fold level of Delta(9)-THC antinociceptive tolerance following chronic treatment with 10mg/kg Delta(9)-THC or 80mg/kg Delta(9)-THC for 6.5 days. Basal and total cytosolic and particulate PKA activities were measured directly in homogenates from the striatum, hippocampus, cerebellum, cortex and lumbar spinal cord. Results from this study indicate that chronic exposure to Delta(9)-THC does not produce an increase in PKA activity in these mouse brain regions or spinal cord. Future work is needed to determine the role of PKA in cannabinoid tolerance in mice.

Aqueous sodium arsenite was administered in doses of 1.0, 1.5 and 2.0 mg/kg body weight (bw)/day by intragastric route to adult Wistar rats, (groups II, III and IV, respectively, n=6 animals/group) for a period of 12 weeks. Controls (group I) received distilled water without added arsenite. At the end of experimental period, four animals from each group were perfusion fixed with 4% paraformaldehyde and two animals from each group were used for fresh tissue purposes (for SDH localization). Fixed tissue blocks from the cervical and lumbar spinal cords were processed for paraffin and cryostat sectioning. Measurements of nuclear area were carried out in cresyl violet (CV) stained paraffin sections by Image Analysis. Cryostat sections (20 microm) of the cervical and lumbar spinal cords of fixed and fresh frozen tissue blocks (n=2/group) were stained for localization of cytochrome oxidase (CO) and succinic dehydrogenase (SDH) activities, respectively. Quantitative estimations of enzyme activities was determined from mean grey values obtained by Image Analysis. The results revealed a dose related reduction in the mean log nuclear area and in the two enzyme activities. The differences noted between the cervical and lumbar motor neurons, viz. (i) the decrease in mean log nuclear area were observed only in cervical motor neurons of animals receiving 2.0 mg/kg bw whereas for the lumbar motor neurons this was true of all doses of arsenite; (ii) in general, CO expression was higher in the cervical than in lumbar spinal cord and (iii) reduction in SDH activity was more pronounced in the cervical spinal cord with doses of 1.5 and 2.0 mg/kg bw as compared to lumbar spinal cord. These observations may throw some light on the earlier observations [Jenkins RB. Inorganic arsenic and the nervous system. Brain 1996;89:479-98] reporting differences in the affliction to arsenic ingestion between the fore limb and hind limb musculature (in human subjects). Our observations ascertain that the neurons, which innervate these musculature, also differ in some characteristics.

In the present study, we investigated the role of phosphorylated calcium/calmodulin-dependent protein kinase-II (pCaMK-II) in nociceptive processing at the spinal and supraspinal levels in the substance P (SP)-induced mouse pain model. In the immunoblot assay, intrathecal (i.t.) injection with SP increased the pCaMK-II level in the spinal cord, and an immunohistochemical study showed that the increase of pCaMK-II immunoreactivity mainly occurred in the laminae I and II areas of the spinal dorsal horn. At the supraspinal level, pCaMK-II was increased in the hippocampus and hypothalamus by i.t. SP injection, and an increase of pCaMK-II immunoreactivity mainly occurred in the pyramidal cells and the stratum lucidum/radiatum layer of the CA3 region of hippocampus and paraventricular nucleus of the hypothalamus. Moreover, pCaMK-II immunoreactivity in the locus coelureus of the brain stem was also increased. The nociceptive behavior induced by SP administered either i.t. or intracerebroventricularly (i.c.v.) was attenuated by KN-93 (a CaMK-II inhibitor). Our results suggest that pCaMK-II located at both spinal cord and supraspinal levels is an important regulator during the nociceptive processes induced by SP administered i.t.

Current work emphasizes that peripheral tissue injury and inflammation results in a heightened sensitivity to subsequent noxious input (hyperalgesia) that is mediated in large part by the spinal synthesis and release of eicosanoids, in particular prostaglandins. Secreted phospholipase A(2)s (sPLA(2)s) form a class of structurally related enzymes that release arachidonic acid from cell membranes that is further processed to produce eicosanoids. We hypothesized that spinal sPLA(2)s may contribute to inflammation-induced hyperalgesia. Spinal cord tissue and cerebrospinal fluid were collected from rats for assessment of sPLA(2) protein expression and sPLA(2) activity. A basal sPLA(2) protein expression and activity was detected in spinal cord homogenate (87+/-17 pmol/min/mg), though no activity could be detected in cisternal cerebrospinal fluid, of naive rats. The sPLA(2) activity did not change in spinal cord tissue or cerebrospinal fluid assessed over 8 h after injection of carrageenan into the hind paw. However, the sPLA(2) activity observed in spinal cord homogenates was suppressed by addition of LY311727, a selective sPLA(2) inhibitor. To determine the role of this spinal sPLA(2) in hyperalgesia, we assessed the effects of lumbar intrathecal (IT) administration of LY311727 in rats with chronic IT catheters in three experimental models of hyperalgesia. IT LY311727 (3-30 microg) dose-dependently prevented intraplantar carrageenan-induced thermal hyperalgesia and formalin-induced flinching, at doses that had no effect on motor function. IT LY311727 also suppressed thermal hyperalgesia induced by IT injection of substance P (30 nmol). Using in vivo spinal microdialysis, we found that IT injection of LY311727 attenuated prostaglandin E(2) release into spinal dialysate otherwise evoked by the IT injection of substance P. Taken together, this work points to a role for constitutive sPLA(2)s in spinal nociceptive processing.

Changes in transport, receptors and production of extracellular adenosine have been observed after induction of hyperthyroidism. Adenosine is associated with inhibitory actions such as reduction in release of excitatory neurotransmitters and antinociception at spinal site. In contrast, ATP acts as an excitatory neurotransmitter and produces pronociceptive actions. ATP may be completely hydrolyzed to adenosine by an enzyme chain constituted by an ATP diphosphohydrolase and an ecto-5'-nucleotidase, as previously described in the spinal cord. Thus, we now investigated the effects of the hyperthyroidism on adenine nucleotide hydrolysis in the spinal cord and verified the nociceptive response in this pathology during different phases of development. Hyperthyroidism was induced in male Wistar rats, aged 5, 60 and 330 days by daily intraperitoneal injections of L-thyroxine (T4) for 14 days. Nociception was assessed with a tail-flick apparatus. Rats starting the treatment aged 5 days demonstrated a significant increase in ADP and AMP hydrolysis and increased tail-flick latency (TFL). In contrast, in the spinal cord from hyperthyroid rats aged 60 and 330 days old, the hydrolysis of ATP, ADP and AMP were significantly decreased. Accordingly, the tail-flick latency was decreased, indicating a hyperalgesic response. These results suggest the involvement of ecto-nucleotidases in the control of the hyperthyroidism-induced nociceptive response in rats at distinct developmental stages.

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3beta-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3beta-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting delta5-3beta-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into delta4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3beta-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3beta-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

Circadian rhythms have been described for numerous transmitter synthesizing enzymes in the brain but rarely in spinal cord. We measured spinal tyrosine-hydroxylase (TH) and nitric oxide synthase (NOS) levels in the thoracic intermediolateral nucleus, the location of sympathetic preganglionic neurons, in male wild type (WT) and dopamine D(3) receptor knockout mice (D(3)KO). TH and NOS levels both displayed circadian patterns in WT and D(3)KO animals with overall reduced TH and increased NOS expression in the D(3)KO mice. The circadian pattern of NOS expression was similar in WT and D(3)KO mice. In contrast, TH expression was inverted in D(3)KO mice, with TH levels consistently lower than in WT throughout the day, but strongly increased temporarily 1 h prior to daylight. TH is the rate-limiting enzyme for the production of dopamine. Spinal dopamine dysfunction is implicated in a sleep disorder called restless legs syndrome (RLS). RLS follows a circadian rhythm and is relieved clinically by dopamine D(3) receptor agonists. Our observations of an altered circadian pattern in spinal dopamine synthesis in D(3)KO animals may provide insight into putative dopaminergic mechanisms contributing to RLS.

This study investigated role of spinal lipoxygenase metabolites in induction of hyperalgesia and development of opioid analgesic tolerance. In the rat, nociception was measured using formalin and tail-flick tests. Intrathecal administration of leukotriene receptor agonist (LTB4) augmented the second phase of the formalin response and marginally increased sensitivity to acute thermal stimulation in the tail-flick test, responses suppressed by 6-(6-(3R-hydroxy-1E,5Z-undecadien-1-yl)-2-pyridinyl)-1,5S-hexanediol (U75302), a leukotriene BLT receptor antagonist. Treatment with 15-hydroxyperoxyeicosatetranoic acid (HPETE) increased phase II formalin activity, but had no effect on tail-flick responses. 12-HPETE failed to produce an effect in either nociceptive test. In the second part of this study, chronic spinal morphine for 5 days produced progressive decline in morphine antinociception and loss in analgesic potency. These effects were attenuated by co-administration of morphine with selective and nonselective lipoxygenase inhibitors. These results suggest involvement of lipoxygenase metabolites in both pain modulation and induction of opioid tolerance at the spinal level.

p21(Cip1/WAF1), known as a cell-cycle inhibitory protein, facilitates neurite outgrowth from neurons when present in the cytoplasm. The molecular mechanism of this action is that p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity. As myelin-derived inhibitors of axonal outgrowth act on neurons by activating Rho, that is responsible for the lack of spontaneous regeneration of the injured central nervous system (CNS), Rho-kinase may be a good molecular target against injuries in the CNS. In this study, we delivered TAT-fusion protein of cytoplasmic p21(Cip1/WAF1) locally after dorsal hemisection of the thoracic spinal cord in rats. The treatment significantly stimulated axonal regeneration and recovery of hindlimb function, and inhibited the cavity formation in the spinal cord after the injury. Cytoplasmic p21(Cip1/WAF1) may provide a potential therapeutic agent that produces functional regeneration following CNS injuries.

We previously demonstrated that spinal protein kinase C (PKC) is involved in the development of a neuropathic pain-like state induced by sciatic nerve ligation, and the morphine-induced rewarding effect is attenuated by sciatic nerve ligation in rodents. Here we first investigated whether sciatic nerve injury could change the activity of a conventional PKC (cPKC) and an atypical PKC isoform PKCzeta in the mouse spinal cord. The second experiment was to investigate whether direct inhibition of spinal PKC by intrathecal (i.t.) administration of a specific PKC inhibitor, 2-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indole-3-yl)maleimide (RO-32-0432), could affect the rewarding effect induced by morphine following sciatic nerve ligation in mice. We found here that the activities of both cPKC and PKCzeta in the spinal cord were clearly increased following sciatic nerve ligation. Furthermore, i.t. administration of RO-32-0432 reversed a long-lasting pain-like syndrome as indicated by thermal hyperalgesia following sciatic nerve ligation in mice. These data provide direct evidence that activated cPKC and PKCzeta in the spinal cord may contribute to the development and maintenance of neuropathic pain. In the present study, we confirmed that the morphine-induced place preference was significantly suppressed by sciatic nerve ligation. It should be mentioned that i.t. pretreatment with RO-32-0432 significantly reversed the attenuation of morphine-induced rewarding effect following sciatic nerve ligation. These results suggest that activation of PKCs, including cPKC and PKCzeta, within the spinal cord is directly responsible for the attenuation of the morphine-induced rewarding effect under a neuropathic pain-like state following sciatic nerve ligation in mice.