Role of reactive oxygen species and spinal cord apoptotic genes in the development of neuropathic pain.

A mouse model of neuropathic pain consisting of chronic constriction injury (CCI) of the sciatic nerve was used to examine the involvement of reactive oxygen species (ROS) in early spinal cord pro-apoptotic gene over-expression during the development of neuropathic pain. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), and caspase-9 in the dorsal horn spinal cord 3 days after chronic constriction injury of sciatic nerve. Consistent with biomolecular data, a marked increase in TUNEL-positive and caspase-3 active form was observed by 3 days CCI. Administration of phenyl-N-tert-butylnitrone (PBN), a potent ROS scavenger, reduced the development of thermal hyperalgesia and mechanical allodynia at 1 and 3 days post-CCI, and decreased the mRNA levels of bax, apaf-1, and caspase-9. PBN also reduced apoptotic and active Caspase-3 positive profiles in the superficial laminae (I-III) of the spinal cord. This study provides evidence that PBN inhibits over-expression of pro-apoptotic genes and neural apoptosis in the spinal cord dorsal horn induced by early-CCI of the sciatic nerve. These findings suggest that ROS regulate expression of some apoptotic genes which might play a role in the onset of neuropathic pain.     

Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn lamina II

We used rats with a sciatic nerve chronic constrictive injury (CCI) and combined behavioural, molecular and morphological approaches to assess the involvement of mGlu5 receptors in neuropathic pain-associated hyperalgesia and spinal cord neuron apoptosis. Mechanical and thermal hyperalgesia developed 2-3 days after surgery. Morphological changes in the ipsilateral L4-L5 lamina II consisted of: (i) cell loss (38 +/- 5%), (ii) increased TUNEL-positive profiles, (iii) decreased SP-immunoreactive primary afferents, and (iv) reactive gliosis. Molecular expression data suggested a bi-phasic response of bcl-2 family genes in CCI. An early (2-3 days post-CCI) E2F1- and p53-independent apoptosis appeared in the spinal cord as the pro-apoptotic bax gene increased (320 +/- 19%), followed by an increased expression of the anti-apoptotic bcl-2 and bcl-xL genes (60 +/- 11% and 110 +/- 15%, respectively) 7 days from CCI. The selective mGlu5 receptor antagonist, MPEP (2 mg/kg i.p. twice daily), prevented the development of thermal hyperalgesia and transiently reduced mechanical hyperalgesia. Despite the MPEP treatment, which normalised bax/bcl-2 and bcl-xL/bcl-xS ratios at all times post-CCI, mechanical hyperalgesia reappeared by 7 days after CCI. Similarly, MPEP was cytoprotective at 3, but not 7 days post-CCI. This study shows that: (a) spinal cord neuron loss may be triggered by a p53- and E2F1-independent apoptosis in lamina II with the participation of glutamate mGlu5 receptors, (b) these receptors seem to be involved transiently, as their blockade was no longer protective by 7 days CCI, and (c) this delayed cell death occurred in the absence of Bax activation, suggesting the involvement of an alternative death pathway.     

鞘内注射氟代柠檬酸对炎性痛敏大鼠的镇痛作用

目的研究鞘内注射氟代柠檬酸(fluorocitrate,Fc)对致炎大鼠痛觉过敏的影响。方法采用大鼠右后爪踝关节外侧皮下注射完全弗氏佐剂(complete freunds adjuvant,CFA)50μl致炎模型。测定给予CFA或Fc前后大鼠机械性缩爪阈值(MWT)和热刺激缩爪潜伏期(TWL)。免疫组化分析脊髓背角星形胶质细胞标记物(GFAP)和小胶质细胞标记物(OX-42)的表达。结果大鼠皮下注射CFA24h后出现明显的炎性痛敏,鞘内注射Fc后4,6,8,10,12h,与CFA组大鼠比较,大鼠MWT明显提高(P<0.01),TWL明显延长(P<0.01)。鞘内注射Fc6h后,降低脊髓背角GFAP和OX-42表达。结论脊髓胶质细胞可能参与炎性痛敏的发生和维持,氟代柠檬酸可能通过抑制其生物活性而发挥镇痛作用。     

JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist

We examined the pharmacological profile of (3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-methoxycyclohexyl) methanone (JNJ16259685). At recombinant rat and human metabotropic glutamate (mGlu) 1a receptors, JNJ16259685 non-competitively inhibited glutamate-induced Ca2+ mobilization with IC50 values of 3.24+/-1.00 and 1.21+/-0.53 nM, respectively, while showing a much lower potency at the rat and human mGlu5a receptor. JNJ16259685 inhibited [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) binding to membranes prepared from cells expressing rat mGlu1a receptors with a Ki of 0.34+/-0.20 nM. JNJ16259685 showed no agonist, antagonist or positive allosteric activity toward rat mGlu2, -3, -4 or -6 receptors at concentrations up to 10 microM and did not bind to AMPA or NMDA receptors, or to a battery of other neurotransmitter receptors, ion channels and transporters. In primary cerebellar cultures, JNJ16259685 inhibited glutamate-mediated inositol phosphate production with an IC50 of 1.73+/-0.40 nM. Subcutaneously administered JNJ16259685 exhibited high potencies in occupying central mGlu1 receptors in the rat cerebellum and thalamus ( ED50=0.040 and 0.014 mg/kg, respectively). These data show that JNJ16259685 is a selective mGlu1 receptor antagonist with excellent potencies in inhibiting mGlu1 receptor function and binding and in occupying the mGlu1 receptor after systemic administration.     

Effects of mGlu1 receptor blockade on anxiety-related behaviour in the rat lick suppression test

Rationale Group I metabotropic glutamate receptor antagonists, which block both the mGlu1 and mGlu5 receptors, have been shown to have anxiolytic effects in the lick suppression test in rats.Objective The anxiolytic potential of the selective mGlu1 antagonist 3,4-dihydro-2H-pyrano[2,3]-quinolin-7-yl)(cis-4-methoxycyclohexyl)methanone (JNJ16259685) was investigated and compared with the mGlu5 antagonist MPEP.Methods Anxiety-related behaviour was assessed in lick suppression and in the elevated zero maze in rats. Non-specific effects on pain threshold, water intake and locomotor activity were also measured.Results Acute administration of JNJ16259685 or MPEP increased the number of licks (lowest active dose 2.5 mg/kg IP for each compound). JNJ16259685 did not increase water intake or reduce acute pain threshold, suggesting that the anxiolytic-like properties are specific. However, acute administration decreased locomotor activity. The effects of chronic administration of JNJ16259685 over 14 days (5 mg/kg bid) on lick suppression were comparable to those seen after acute administration, arguing against development of behavioural tolerance or sensitisation. Yet, there was a tendency for an increase in locomotor activity after cessation of chronic treatment. Acute co-administration of both JNJ16259685 and MPEP had additive effects on the number of licks. No anxiolytic-like properties of JNJ16259685 were observed in the elevated zero maze.Conclusion Our data suggest that the anxiolytic-like effects induced by group I metabotropic glutamate receptor antagonists are mediated through both mGlu1 and mGlu5 receptors. Rather than producing a general anxiolytic-like effect, the effects seen following mGlu1 antagonism seem task-dependent, as prominent effects were seen in a conflict procedure, but not in a task based on spontaneous exploration.     

星形胶质细胞在坐骨神经分支选择性结扎神经病理性痛中的作用

目的 探讨星形胶质细胞在坐骨神经分支选择性结扎(spared nerve injury,SNI)神经病理性痛中的作用.方法 24只SD大鼠随机分为4组(6只/组):SNI组(建立SNI动物模型同时行鞘内置管术,术后13 d鞘内给予生理盐水);假手术组(处理方式同SNI组,但不损伤坐骨神经及其分支);L-α-aminoadipate(LAA)组(制作SNI动物模型同时行鞘内置管,术后13 d鞘内给予LAA);对照组(不给予任何处理因素).术前2 d及术后14 d检测机械痛和神经病理性痛阈值,术后14 d以Real-time PCR法检测脊髓背角GFAP mRNA变化.结果 术前2 d各组大鼠机械痛域及热痛阈无明显差异(P>0.05).术后14 d假手术组机械痛域、热痛阈及脊髓背角GFAP mRNA较对照组均无明显变化(P>0.05);与对照组相比,SNI组机械痛域及热痛阈明显降低(P<0.05),脊髓背角GFAP mRNA明显增高(P<0.05);与SNI组相比,LAA组机械痛域及热痛阈均明显增高(P<0.05),脊髓背角GFAP mRNA明显降低(P<0.05).结论 LAA特异性抑制脊髓背角星形胶质细胞活性可缓解SNI大鼠神经病理性痛,提示脊髓背角星形胶质细胞活化是SNI大鼠神经病理性的重要机制.     

mGlu and NMDA receptor contributions to capsaicin-induced thermal and mechanical hypersensitivity

Metabotropic glutamate (mGlu) receptors are G protein-coupled receptors, some of which are localized in the spinal cord dorsal horn, and are involved with pain perception. The anti-nociceptive effects of intrathecal (i.t.) pretreatment with various mGlu receptor agonists and antagonists were assessed in Long Evans rats with mechanical and thermal hypersensitivity after sub-dermal injection of capsaicin in the hindpaw. Selective group II (aminopyrrolidine-2R,4R-dicarboxylate, APDC) and group III (l-2-amino-4-phosphonobutyrate, L-AP4) agonists, as well as selective mGlu(1) (1-aminoindan-1,5(R,S)-dicarboxylic acid, AIDA) and mGlu(5) (2-methyl-6-(phenylethynyl)-pyridine, MPEP) receptor subtype antagonists were compared with that of an NMDA receptor antagonist (dizocilipine maleate, MK-801). The rats were observed for signs of capsaicin-induced mechanical and thermal hypersensitivity 15 min after capsaicin injection, and 20 min following i.t. drug administration. Results indicate there was a dose-dependent reduction in capsaicin-induced mechanical hypersensitivity for all mGlu receptor agents; with maximal increases in mechanical thresholds that were 7-fold for AIDA and APDC, 7.5-fold for L-AP4 and 5.6-fold for MPEP. However, only a weak reduction (often non-significant) in thermal hypersensitivity was observed with each of the mGlu receptor drugs; thermal latencies were maximally increased by 125% (AIDA), 0% (MPEP), 8% APDC and 205% (L-AP4). By contrast, the highest dose of MK-801 was able to significantly reduce both mechanical (maximal 6.67-fold increase in threshold) and thermal (maximal 3-fold increase in latencies) hyperalgesia. We conclude that mGlu receptors contribute to the development of mechanical allodynia, but not thermal hyperalgesia, following capsaicin injury; while iGluRs may contribute to both thermal and mechanical hypersensitivity.     

Activation of group I mGlu receptors contributes to facilitation of NMDA receptor membrane current in spinal dorsal horn neurons after hind paw inflammation in rats

The interaction between the group I metabotropic glutamate (mGlu) receptors and N-methyl-D-aspartate (NMDA) receptors plays a critical role in spinal hyperexcitability and hyperalgesia. The cellular mechanisms underlying this interaction remain unknown. Utilizing an ex vivo spinal slice preparation from young adult rats, we investigated the group I mGlu receptor modulation of NMDA receptor-mediated current in superficial dorsal horn neurons by patch clamp recording after complete Freund's adjuvant (CFA)-induced hind paw inflammation. We show that NMDA receptor-mediated dorsal root stimulation-evoked EPSC (eEPSC) and NMDA-induced current was enhanced in the inflamed rats, compared to na?ve rats and this effect was attenuated by AIDA (1 mM), a group I mGlu receptor antagonist. There were also increases in the frequency and amplitude of miniature excitatory postsynaptic currents in the presence of tetrodotoxin, suggesting enhanced presynaptic glutamate release probability and postsynaptic membrane responsiveness in inflamed rats. DHPG (10 μM), a selective group I mGlu receptor agonist, further facilitated NMDA receptor-mediated eEPSC and NMDA-induced current in inflamed rats. The DHPG-produced facilitation of NMDA-induced current was blocked by intracellular dialysis of GDP-beta-S (1 mM), a G protein antagonist, and BAPTA (15 mM), an intracellular calcium chelating agent; and by pretreatment with U73,122 (10 μM), a PLC inhibitor, or 2-APB (100 μM), an IP?-receptor antagonist. These findings support the hypothesis that signal transduction coupling between group I mGlu receptors and NMDA receptors underlies the activation of NMDA receptors in spinal hyperexcitability and hyperalgesia.     

JNJ16259685, a selective mGlu1 antagonist, suppresses isolation-induced aggression in male mice

mGlu1 receptors are present in brain regions involved in aggression modulation. This study examines the effects of 3-4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl-(cis-4-methoxycyclohexyl)-methanone (JNJ16259685; 0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/kg, i.p), a selective antagonist of the mGlu1 receptors, on agonistic interactions between male mice. Individually housed mice were exposed to anosmic "standard opponents" 30 min after drug administration. Ten minutes of diadic interactions was staged between a singly housed and an anosmic mouse in a neutral area. The encounters were videotaped and the accumulated time allocated by subjects to ten broad behavioural categories was estimated using an ethologically based analysis. JNJ16259685 (all doses) produced a significant reduction of offensive behaviours (threat and attack), without affecting immobility. These findings suggest for the first time a role for mGlu1 receptors in aggression regulation.     

Potent and specific action of the mGlu1 antagonists YM-298198 and JNJ16259685 on synaptic transmission in rat cerebellar slices

BACKGROUND AND PURPOSE: Specific and selective inhibitors for mGlu1 receptors are presently inadequate. A new generation of non-competitive mGlu1 antagonists with low nanomolar potencies is emerging. We evaluated two new compounds, YM-298198 and JNJ16259685, for effectiveness, potency and specificity for the first time in a brain slice preparation. EXPERIMENTAL APPROACH: Patch-clamp recording of Purkinje neurones in cerebellar slices were obtained. The slow mGlu1-mediated EPSP was used to establish a concentration-response curve. Fast excitatory synaptic inputs were tested for non-specific effects. KEY RESULTS: YM-298198 and JNJ16259685 inhibited the synaptic activation of mGlu1 in a concentration-dependent manner (IC(50) values of 24 nM and 19 nM, respectively). The antagonists were slow to inhibit and to reverse on washout, probably due to their lipophilic nature. There were no non-specific effects on fast AMPA receptor-mediated synaptic transmission in the cerebellum. CONCLUSIONS AND IMPLICATIONS: These compounds are more than a thousand-fold more potent than previously available compounds. Their selectivity and specificity will be very useful for studying the role of mGlu1 receptors both in vitro and in vivo.     

Synaptic mechanisms in nociception: emerging targets for centrally-acting analgesics

The lifetime incidence of chronic pain in Western populations is almost 50%, but current pharmacological treatments are poorly tolerated and ineffective against some types of pain, giving rise to a demand for new analgesic drugs. The primary symptoms of chronic pain are allodynia, hyperalgesia and spontaneous pain, all of which indicate a high level of excitability in central pain processing systems. Recent basic research has identified a bewildering number of molecules as actual or putative mediators of spinal hyperexcitability, and the main thrust of the present article is that antagonists for these molecules should provide new antihyperalgesic or analgesic treatments. Ionotropic glutamate receptors (particularly N-methyl-D-aspartate [NMDA], but including other calcium-permeable receptors), in conjunction with voltage-gated calcium channels, permit ingress of calcium ions into spinal dorsal horn neurones. In addition, group I metabotropic glutamate (mGlu) and tachykinin NK₁ and NK₃ receptors give rise to release of calcium from intracellular stores. In theory, antagonists for any of these receptors ought to be antihyperalgesic, but recent experience in clinical trials with NK₁ receptor blockers has been disappointing. More encouragingly, non-selective antagonists for glutamate NMDA receptors are effective in all types of chronic pain in humans, albeit with side effects that are unacceptable. The recognition that NMDA receptors exist in multiple subtypes, and that the NR2B subtype is found (among only a few other sites) in the superficial dorsal horn of the spinal cord in rat, suggests that selective antagonists for these receptors may offer analgesia without the attendant problems of non-selective drugs. Selective antagonists for other calcium-permeable glutamate receptors, mGlu group I, NK₃ and CGRP1 receptors may also have a future as antihyperalgesic agents, but all are at a very early stage of development. At present, the best hope for a powerful broad-spectrum antihyperalgesic lies with the NMDA receptor subtypes.     

Activation of spinal orexin-1 receptor produces anti-allodynic effect in the rat carrageenan test

Orexin-A and orexin-1 receptors are found in the dorsal root ganglion cells and the spinal dorsal horn and this suggests that orexin-A is involved in the spinal nociceptive transmission. The authors examined the effect of intrathecally administered orexin-A on the level of mechanical allodynia and thermal hyperalgesia induced by paw carrageenan injection in the rat. Intrathecal injection of 0.3 and 3 nmol of orexin-A suppressed the level of mechanical allodynia, but not that of thermal hyperalgesia, and the effect of orexin-A on mechanical allodynia was antagonized by the pretreatment of 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride, SB-334867, a selective orexin-1 receptor antagonist. These data suggest that the activation of spinal orexin-1 receptor modulates the mechanical information transmission, but not thermal information transmission, in the spinal cord during carrageenan test.     

Two new non-competitive mGlu1 receptor antagonists are potent tools to unravel functions of this mGlu receptor subtype

The validation of the selective, potent and systemically active non-competitive mGlu1 antagonists YM-298198 and JNJ16259685 in a physiological functional assay will facilitate elucidation of this receptor's role in brain function and as a potential drug target.     

Antisense oligonucleotide knockdown of mGluR1 alleviates hyperalgesia and allodynia associated with chronic inflammation

Chronic inflammation induced by injection of complete Freund's adjuvant (CFA) into one hindpaw elicits thermal hyperalgesia and mechanical allodynia in the injected paw. Metabotropic glutamate receptors (mGluRs) have been implicated in dorsal horn neuronal nociceptive responses and pain associated with short-term inflammation. The goal of the present study was to assess the role of mGluR1 in the hyperalgesia and allodynia associated with the CFA model of chronic inflammation. Here we show that antisense (AS) oligonucleotide knockdown of spinal mGluR1 attenuates thermal hyperalgesia and mechanical allodynia in rats injected with CFA in one hindpaw. When intrathecal infusion of mGluR1 AS oligonucleotide (50 microg/day) began prior to CFA injection, mechanical allodynia was attenuated from Days 1 to 8 following CFA injection, whereas heat hyperalgesia was attenuated on Day 1 and then from Days 4 to 8. When intrathecal infusion of mGluR1 AS oligonucleotide was begun 2 days after CFA injection, both mechanical allodynia and heat hyperalgesia were attenuated at all time points following the oligonucleotide infusion. Thus, the present data suggest a role for mGluR1 in persistent inflammatory nociception.     

The challenge of chronic pain

Chronic pain is a complex problem with staggering negative health and economic consequences. The complexity of chronic pain is presented within Cervero and Laird's model that describes three phases of pain, including pain without tissue damage, pain with tissue damage and inflammation, and neuropathic pain. The increased afferent input in phases 2 and 3 of chronic pain produces marked changes in primary afferents, dorsal root ganglia, and spinal cord dorsal horn. These changes promote the symptoms of chronic pain, including spontaneous pain, hyperalgesia, and allodynia. Increased afferent input also evokes supraspinal input to the dorsal horn, including biphasic innervation from the ventromedial medulla and A7 catecholamine cell group, that promotes hyperalgesia and allodynia. More rostral brain structures, such as the lateral hypothalamus, amygdala, and hippocampus, may also play a role in chronic pain. Although much has been discovered about the multiple pathological mechanisms involved in chronic pain, further research is needed to fully comprehend these mechanisms.     

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against β-amyloid toxicity

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with β-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17β-estradiol (17βE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17βE2. The selective ERβ agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17βΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17βΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17βE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.     

蛛网膜下隙注射胶质细胞源性神经营养因子抑制脊神经结扎大鼠脊髓背角胶质原纤维酸性蛋白表达

目的观察蛛网膜下隙注射胶质细胞源性神经营养因子(GDNF)对脊神经结扎(SNL)大鼠脊髓背角胶质原纤维酸性蛋白(GFAP)的影响。方法采用结扎SD大鼠第5~第6腰脊神经(L5~L6)制备SNL模型。术后隔日一次性蛛网膜下隙注射10μl GDNF2 g·L-1组。术后第3,7和14天采用免疫组织化学和Western免疫印迹法测定脊髓背角处GFAP蛋白表达。结果免疫组化结果显示,SNL组在术后第3天、第7天和第14天脊髓GFAP阳性细胞数目明显增加,可持续至第14天,而正常对照组与假手术组的星形胶质细胞未发生此种改变。GDNF组的阳性细胞显著减少。Western印迹结果显示,正常对照组和假手术组脊髓背角均出现GFAP免疫阳性条带,灰度值较低;SNL在术后第3天即可诱导脊髓背角GFAP蛋白的表达,随着时间的延长,GFAP蛋白的灰度值逐渐升高,术后第3,7,14天分别为2.55±0.33,2.88±0.79和3.12±0.75(P<0.01)。与SNL模型组比较,GDNF可显著降低GFAP的表达水平,术后第3,7,14天分别为1.61±0.38,1.65±0.64和1.57±0.41(P<0.01)。结论蛛网膜下隙注射GDNF减轻大鼠神经病理性疼痛机制可能与其抑制脊髓背角GFAP蛋白表达有关。     

Spinal nerve ligation reduces nitric oxide synthase activity and expression: effect of resveratrol

The effect of resveratrol on activity and expression of nitric oxide synthase (NOS) in the spinal cord of neuropathic rats was assessed. Spinal nerve ligation produced tactile allodynia along with a reduction of catalytic activity of the constitutive Ca²⁺-dependent NOS (eNOS and nNOS isoforms) in the ipsilateral dorsal horn, but not contralateral dorsal or ipsilateral or contralateral ventral, spinal cord at 1, 5, 10 and 15 days after surgery compared to naïve and sham-operated animals. Nerve ligation also induced a reduction of nNOS expression in the ipsilateral dorsal horn spinal cord at 10 and 15 days after surgery. Intrathecal resveratrol reduced allodynia and reversed the reduction of constitutive Ca²⁺-dependent NOS activity in the ipsilateral dorsal spinal cord. Moreover, resveratrol significantly reversed the reduction of nNOS expression in the ipsilateral dorsal horn spinal cord. Results show that spinal nerve ligation leads to development of tactile allodynia along with a reduction in constitutive Ca²⁺-dependent NOS activity and nNOS isoform expression in the ipsilateral dorsal horn. Data suggest that resveratrol may reduce tactile allodynia in neuropathic rats by restoring altered NOS activity and expression.     

Spinal nerve ligation reduces nitric oxide synthase activity and expression: Effect of resveratrol

The effect of resveratrol on activity and expression of nitric oxide synthase (NOS) in the spinal cord of neuropathic rats was assessed. Spinal nerve ligation produced tactile allodynia along with a reduction of catalytic activity of the constitutive Ca²⁺-dependent NOS (eNOS and nNOS isoforms) in the ipsilateral dorsal horn, but not contralateral dorsal or ipsilateral or contralateral ventral, spinal cord at 1, 5, 10 and 15 days after surgery compared to naïve and sham-operated animals. Nerve ligation also induced a reduction of nNOS expression in the ipsilateral dorsal horn spinal cord at 10 and 15 days after surgery. Intrathecal resveratrol reduced allodynia and reversed the reduction of constitutive Ca²⁺-dependent NOS activity in the ipsilateral dorsal spinal cord. Moreover, resveratrol significantly reversed the reduction of nNOS expression in the ipsilateral dorsal horn spinal cord. Results show that spinal nerve ligation leads to development of tactile allodynia along with a reduction in constitutive Ca²⁺-dependent NOS activity and nNOS isoform expression in the ipsilateral dorsal horn. Data suggest that resveratrol may reduce tactile allodynia in neuropathic rats by restoring altered NOS activity and expression.     

Activation of ERK／CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats

Aim: To investigate whether activation and translocation of extracellular signalregulated kinase (ERK) is involved in the induction and maintenance of neuropathic pain, and effects of activation and translocation of ERK on expression of pCREB and Fos in the chronic neuropathic pain. Methods: Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica‘s trifurcation at approximately 1.0 mm intervals with 4-0 silk sutures. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or phosphorothioate-modified antisense oligonucleotides (ODN) were intrathecally administered every 12 h, 1 d pre-chronic constriction injury (CCI) and 3 d post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The expression of pERK, pCREB, and Fos were assessed by both Western blotting and immunohistochemical analysis. Results: Intrathecal injection of U0126 or ERK antisense ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of p-ERK-IR neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK expression showed that the levels of both cytosol and nuclear pERK, but not total ERK, were increased at all points after CCI and reached a peak level on postoperative d 5. CCI also significantly increased the expression of pCREB and Fos. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord and Fos was expressed in laminae I and II of the ipsilateral spinal dorsal horn. Intrathecal injection of U0126 or ERK antisense ODN markedly suppressed the increase of CCI-induced pERK, pCREB and c-Fos expression in the spinal cord. Conclusion:The activation of ERK pathways contributes to neuropathic pain in CCI rats, and the function of pERK may partly be accomplished via the cAMP response element binding protein (CREB)-dependent gene expression.