Rosuvastatin Synergistically Enhances the Antinociceptive Efficacy of Duloxetine in Paclitaxel-Induced Neuropathic Pain in Mice.

Paclitaxel, a widely used cancer chemotherapeutic agent, has high incidence of neurotoxicity associated with the production of neuropathic pain, for which only duloxetine has shown significant but moderate analgesic effect. Since statins, classically used to reduce hypercholesterolemia, have shown antinociceptive effect in preclinical studies on neuropathic pain, we studied whether the antinociceptive efficacy of duloxetine could be synergistically potentiated by rosuvastatin in a model of paclitaxel-induced neuropathy in mice. The astrocytic and microglial responses in the spinal cord of paclitaxel-treated mice were also assessed by measuring GFAP and CD11b proteins, respectively. Paclitaxel treatment did not impair motor coordination and balance in rotarod testing. Rosuvastatin, duloxetine, and the rosuvastatin/duloxetine combination (combined at equieffective doses) dose-dependently decreased mechanical allodynia (ED30, von Frey testing) and thermal hyperalgesia (ED50, hot plate testing) in paclitaxel-treated mice. Isobolographic analysis showed a superadditive interaction for rosuvastatin and duloxetine, as both the ED30 and ED50 for the rosuvastatin/duloxetine combination contained only a quarter of each drug compared to the individual drugs. The rosuvastatin/duloxetine combination reversed paclitaxel-induced GFAP overexpression, indicating that such effects might depend in part on astrocyte inactivation. Results suggest that statins could be useful in synergistically enhancing the efficacy of duloxetine in some chemotherapy-induced neuropathic conditions.

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 µM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 µM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 µM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.

Magnesium Salt, a Simple Strategy to Improve Methadone Analgesia in Chronic Pain: An Isobolographic Preclinical Study in Neuropathic Mice.

Analgesic efficacy of methadone in cancer and chronic non-cancer pains is greater than that of other opioids, probably because of its unique pharmacokinetics properties and also because it targets glutamatergic receptors in addition to µ-opioid receptors. However, methadone has drawbacks which are clearly related to dosing and treatment duration. The authors hypothesized that the antinociceptive efficacy of methadone could be synergistically potentiated by magnesium and copper salts in a preclinical mouse model of chronic pain, using the intraplantar formalin test as algesimetric tool. The spared nerve injury mice model was used to generate mononeuropathy. A low dose (0.25%) formalin was injected in the neuropathic limb in order to give rise only to Phase I response, resulting from direct activation by formalin of nociceptive primary afferents. Licking/biting of the formalin-injected limb was evaluated as nociceptive behavior during a 35-min observation period. Dose-response curves for intraperitoneal magnesium sulfate (10, 30, 100, and 300 mg/kg i.p.), copper sulfate (0.1, 0.3, 1, and 3 mg/kg i.p.) and methadone (0.1, 0.3, 1, and 3 mg/kg i.p.) allowed to combine them in equieffective doses and to determine their interaction by isobolographic analysis. Magnesium sulfate, copper sulfate and methadone dose-dependently decreased the nociceptive response evoked by formalin injection, the respective ED50 being 76.38, 1.18, and 0.50 mg/kg i.p. Isobolographic analysis showed a superadditive interaction for magnesium and methadone. Indeed, despite that both ED50 are obviously equieffective, the ED50 for the MgSO4/methadone combination contained less than one third of the methadone having the ED50 for methadone alone. For the CuSO4/methadone combination, the interaction was only additive. Extrapolated to clinical settings, the results suggest that magnesium salts might be used to improve synergistically the efficacy of methadone in neuropathy, which would allow to reduce the dose of methadone and its associated side effects.

A pH-responsive nanoparticle targets the neurokinin 1 receptor in endosomes to prevent chronic pain.

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NK1R) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NK1R in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NK1R-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NK1R antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.

The antinociceptive effect of resveratrol in bone cancer pain is inhibited by the Silent Information Regulator 1 inhibitor selisistat.

OBJECTIVES: To study the antinociceptive effect of single and repeated doses of resveratrol in a bone cancer pain model, and whether this effect is prevented by the Silent Information Regulator 1 (SIRT1) inhibitor selisistat. METHODS: The femoral intercondylar bone of BALB/c mice was injected with 1 000 000 BJ3Z cancer cells. Bone resorption and tumour mass growth (measured by in vivo X-ray and fluorescence imaging), as well as mechanical nociceptive thresholds (von Frey device) and dynamic functionality (rotarod machine), were evaluated during the following 4 weeks. Acute resveratrol (100 mg/kg i.p.) and/or selisistat (10 mg/kg s.c.) were administered on day 14. Chronic resveratrol (100 mg/kg i.p., daily) and/or selisistat (0.5 µg/h s.c., Alzet pump) were administered between days 14 and 20. KEY FINDINGS: Tumour growth gradually incremented until day 31, while mechanical hyperalgesia started on day 3 after cancer cell injection. Acute resveratrol increased the mechanical threshold of pain (peaking at 1.5 h), while the dynamic functionality decreased. Chronic resveratrol produced a sustained antinociceptive effect on mechanical hyperalgesia and improved the loss of dynamic functionality induced by the bone cancer tumour. Selisistat prevented all the effects of resveratrol. CONCLUSIONS: Acute and chronic resveratrol induces antinociceptive effect in the model of metastatic osseous oncological pain, an effect that would be mediated by SIRT1 molecular signalling.

Burst-Like Subcutaneous Electrical Stimulation Induces BDNF-Mediated, Cyclotraxin B-Sensitive Central Sensitization in Rat Spinal Cord.

Intrathecal administration of brain derived neurotrophic factor (BDNF) induces long-term potentiation (LTP) and generates long-lasting central sensitization in spinal cord thus mimicking chronic pain, but the relevance of these observations to chronic pain mechanisms is uncertain. Since C-fiber activation by a high-frequency subcutaneous electrical stimulation (SES) protocol causes spinal release of BDNF and induces spinal cord LTP, we propose that application of such protocol would be a sufficient condition for generating long-lasting BDNF-mediated central sensitization. Results showed that application of burst-like SES to rat toes produced (i) rapid induction of hyperalgesia that lasted for more than 3 weeks, (ii) early increase of C-reflex activity followed by increased wind-up scores lasting for more than 1 week, and (iii) early increase followed by late decrease in BDNF protein levels and phosphorylated TrkB that lasted for more than 1 week. These changes were prevented by the TrkB antagonist cyclotraxin-B administered shortly before SES, while hyperalgesia was reversed by cyclotraxin-B administered 3 days after SES. Results suggest that mechanisms underlying central sensitization first involve BDNF release of probably neuronal origin, followed by brief increased expression of likely glial BDNF and pTrkB that could switch early phase sensitization into late one.

Antinociception Induced by Copper Salt Revisited: Interaction with Ketamine in Formalin-Induced Intraplantar and Orofacial Pain in Mice.

AIMS: To evaluate in mice the antinociceptive effect of copper in spinal and trigeminal nociceptive pathways by using the intraplantar and orofacial formalin tests, respectively, and to examine whether this effect may interact synergistically with ketamine-induced antinociception. METHODS: Nociceptive behaviors (licking/biting of the formalin-injected limb and rubbing/scratching of the formalin-injected orofacial area) in male mice were evaluated during a 45-minute observation period post-formalin injection. Dose-response curves for intraperitoneal (ip) copper sulfate and ketamine allowed their combination in equi-effective doses, and their interaction was determined with isobolographic analysis. The results were examined with one-way analysis of variance followed by the Bonferroni post hoc test. Significance was accepted at an alpha level of .05. RESULTS: Irrespective of the region injected with formalin (upper lip or hindlimb), copper sulfate (0.3, 1.0, and 3.0 mg/kg) and ketamine (1.0, 3.0, and 10 mg/kg) dose-dependently decreased the nociceptive behaviors evoked by formalin injection. Isobolographic analysis showed a superadditive interaction between copper and ketamine at the spinal level, but this interaction was only additive at the trigeminal level. CONCLUSION: The results suggest that copper salts could be used to synergistically improve the efficacy of some commercial centrally acting analgesic agents, such as ketamine, while reducing the possibility of side effects. However, a synergistic effect probably should not be expected if treatment is for orofacial pain.

Caracterización del uso de metadona en pacientes paliativos en el Instituto Nacional del Cáncer, Santiago de Chile / Characterization of methadone use in palliative patients at the National Cancer Institute, Santiago, Chile

OBJETIVOS: realizar un estudio de tipo descriptivo y retrospectivo para caracterizar el uso y efecto de metadona en una población de pacientes de la unidad de cuidados paliativos del Instituto Nacional del Cáncer. MATERIALES Y MÉTODOS: los datos analizados se obtuvieron desde los registros de la farmacia y las fichas clínicas de los pacientes de la unidad de cuidados paliativos del Instituto Nacional del Cáncer que estaban siendo tratados con metadona durante el mes de agosto de 2013. las variables cuantificadas fueron edad, sexo, diagnóstico oncológico, tipo de dolor, motivo de indicación de metadona, duración del tratamiento, dosis utilizadas, respuesta analgésica y uso concomitante con analgésicos no opioides. RESULTADOS: la población de pacientes bajo control mensual en la unidad de cuidados paliativos al mes de agosto fue de 445 pacientes. en el estudio se incluyeron a 31 pacientes que estaban en ese periodo tratados con metadona, lo que representa un 7% del total de pacientes. la indicación, según tipo de dolor, fue en un 80,6% por dolor de tipo neuropático o mixto. la indicación de metadona se debió, en un 68%, a una rotación de opioides y solo en un 3% fue por indicación primaria. la dosis promedio diaria fluctuó entre 16,7 mg, al inicio del tratamiento; y 26,1 mg, al momento del estudio o periodo de observación. la mediana de uso fue de 211 días. la intensidad del dolor, medida por la escala numérica verbal, fue de 8,3 ± 0,3 mg al inicio del tratamiento y 5,4 ± 0,6 mg durante el control de agosto de 2013, lo que significa una disminución promedio de 34,9%. DISCUSIÓN: el uso de metadona en la unidad de cuidados paliativos del Instituto Nacional del Cáncer está en concordancia con las propuestas internacionales, indicándose principalmente como rotación de opioides y en el tratamiento del dolor neuropático. el análisis de las fichas mostró utilización de dosis bajas de metadona (menores de 30 mg), con pequeños incrementos de dosis durante el período de tratamiento, obteniéndose como resultado una reducción significativa del dolor.

OBJETIVES: to carry out a descriptive and retrospective study to characterize the use and effect of methadone in a group of patients of the Palliative Care Unit of the National Cancer Institute. MATERIALS AND METHODS: the information was obtained from the patient pharmacy and clinical records at the palliative care unit of the National Cancer Institute who were being treated with methadone during the month of august, 2013. the variables assessed were age, sex, oncology diagnosis, type of pain, reason for methadone prescription, treatment duration, dosage, pain response and associated use of non- opioid analgesics. RESULTS: the patient group under monthly monitoring at the palliative care unit in august was made up of 445 people. the study included 31 patients who at that time were being treated with methadone, 7% of the total. the indication according to the type of pain was in an 80.6% caused by a neuropathic or mixed pain. a 68% of the methadone indication was caused by an opioid rotation and only a 3% by a primary indication. the average daily dose went from 16.7 mg at the beginning to 26.1 mg at the moment the study or observation period was carried out. the use average was of 211 days. The pain intensity, using a numeric scale, was of 8.3 ± 0.3 mg at the beginning of the treatment and a 5.4 ± 0.6 mg during the august 2013 control, which means an average decrease of 34,9 %. DISCUSSION: the use of methadone at the palliative care unit of the National Cancer Institute is in accordance with the international proposals, being indicated mostly as opioid rotation and in the neuropathic pain treatment. the record analysis showed a low dose use of methadone (lower than 30 mg), with small dose increase during the treatment period, getting a significant pain decrease as a result

Pannexin 1: a novel participant in neuropathic pain signaling in the rat spinal cord.

Pannexin 1 (panx1) is a large-pore membrane channel expressed in many tissues of mammals, including neurons and glial cells. Panx1 channels are highly permeable to calcium and adenosine triphosphatase (ATP); on the other hand, they can be opened by ATP and glutamate, two crucial molecules for acute and chronic pain signaling in the spinal cord dorsal horn, thus suggesting that panx1 could be a key component for the generation of central sensitization during persistent pain. In this study, we examined the effect of three panx1 blockers, namely, 10panx peptide, carbenoxolone, and probenecid, on C-reflex wind-up activity and mechanical nociceptive behavior in a spared nerve injury neuropathic rat model involving sural nerve transection. In addition, the expression of panx1 protein in the dorsal horn of the ipsilateral lumbar spinal cord was measured in sural nerve-transected and sham-operated control rats. Sural nerve transection resulted in a lower threshold for C-reflex activation by electric stimulation of the injured hindpaw, together with persistent mechanical hypersensitivity to pressure stimuli applied to the paw. Intrathecal administration of the panx1 blockers significantly depressed the spinal C-reflex wind-up activity in both neuropathic and sham control rats, and decreased mechanical hyperalgesia in neuropathic rats without affecting the nociceptive threshold in sham animals. Western blotting showed that panx1 was similarly expressed in the dorsal horn of lumbar spinal cord from neuropathic and sham rats. The present results constitute the first evidence that panx1 channels play a significant role in the mechanisms underlying central sensitization in neuropathic pain.

Stage-dependent C-reflex, pain-like behavior and opioid analgesia during the induction of chronic arthritis in rats.

Chronic arthritis (CA) is a common clinical entity associated with persistent pain and limited response to opioid analgesic therapy. However, it is unknown whether these features of CA change depending on its stage of evolution. To address this, in a well-established animal model of CA we studied the time course of electromyographic responses to electrical stimulation of C fibers (C-reflex), pain-like behavior as a response to mechanical nociceptive stimulation, and the inhibition of both responses by a prototypic opioid analgesic, morphine. To induce CA, rats received a single injection of complete Freund's adjuvant into the ankle joint and the C-reflex responses to electrical stimuli or the nociceptive response to paw pressure test were studied 2, 4 or 6 weeks later. The C-reflexes evoked by threshold and supra-threshold electrical stimulation exhibited progressive increases together with enhancement of the nociceptive behavior to mechanical stimulation during induction of monoarthritis. Notably, while systemic morphine produced antinociceptive effects upon both experimental approaches, the effects were markedly reduced during the early stages of CA but enhanced at later stages. These data indicate that C-reflex and pain-like responses evolve in parallel, and are inhibited by morphine in a stage-dependent manner through the induction of CA. The present results may contribute to explain the enhanced pain response and variable analgesic efficacy of opioids that characterize arthritic pain in humans.

Disruption of 5-HT2A receptor-PDZ protein interactions alleviates mechanical hypersensitivity in carrageenan-induced inflammation in rats.

Despite common pathophysiological mechanisms, inflammatory and neuropathic pain do not respond equally to the analgesic effect of antidepressants, except for selective serotonin reuptake inhibitors (SSRIs), which show a limited efficacy in both conditions. We previously demonstrated that an interfering peptide (TAT-2ASCV) disrupting the interaction between 5-HT2A receptors and its associated PDZ proteins (e.g. PSD-95) reveals a 5-HT2A receptor-mediated anti-hyperalgesic effect and enhances the efficacy of fluoxetine (a SSRI) in diabetic neuropathic pain conditions in rats. Here, we have examined whether the same strategy would be useful to treat inflammatory pain. Sub-chronic inflammatory pain was induced by injecting λ-carrageenan (100 µl, 2%) into the left hind paw of the rat. Mechanical hyperalgesia was assessed after acute treatment with TAT-2ASCV or/and fluoxetine (SSRI) 2.5 h after λ-carrageenan injection. Possible changes in the level of 5-HT2A receptors and its associated PDZ protein PSD-95 upon inflammation induction were quantified by Western blotting in dorsal horn spinal cord. Administration of TAT-2ASCV peptide (100 ng/rat, intrathecally) but not fluoxetine (10 mg/kg, intraperitoneally) relieves mechanical hyperalgesia (paw pressure test) in inflamed rats. This anti-hyperalgesic effect involves spinal 5-HT2A receptors and GABAergic interneurons as it is abolished by a 5-HT2A antagonist (M100907, 150 ng/rat, intrathecally) and a GABAA antagonist, (bicuculline, 3 µg/rat, intrathecally). We also found a decreased expression of 5-HT2A receptors in the dorsal spinal cord of inflamed animals which could not be rescued by TAT-2ASCV injection, while the amount of PSD-95 was not affected by inflammatory pain. Finally, the coadministration of fluoxetine does not further enhance the anti-hyperalgesic effect of TAT-2ASCV peptide. This study reveals a role of the interactions between 5-HT2A receptors and PDZ proteins in the pathophysiological pathways of inflammatory pain and opens new perspectives in its control thanks to molecules disrupting 5-HT2A receptor/PDZ protein interactions.

Antinociceptive interaction of (±)-CPP and propentofylline in monoarthritic rats.

INTRODUCTION: Multiple studies have shown that glial cells of the spinal cord, such as astrocytes and microglia, have close contact with neurons, suggesting the term tripartite synapse. In these synapses, astrocytes surrounding neurons contribute to neuronal excitability and synaptic transmission, thereby increasing nociception and thus the persistence of chronic pain. Conversely, the N-methyl-D-aspartate (NMDA) receptor is crucial in the generation and maintenance of chronic pain. It has multiple sites of modulation. One is the site of recognition of extracellular neurotransmitter (glutamate), which can be blocked by competitive antagonists such as (3-(2-carboxipiperazin-4)1-propyl phosphonic acid), (±)-CPP, resulting in a blockade of the calcium current and thus the intracellular transduction process. In the present study, we investigated whether the potential antinociceptive effect of glial inhibition produced by propentofylline (PPF) can be enhanced when combined with an NMDA-receptor inhibitor such as (±)-CPP. METHODS: We used Sprague-Dawley monoarthritic rats. The monoarthritis was induced by injection of complete Freund adjuvant in the right tibiotarsal joint. Four weeks later, rats were treated with PPF (1, 10, 30, and 100 µg/10 µl) intrathecally (i.t.) for 10 days, injected once with (±)-CPP (2.5, 5, 12.5, 25, 50, and 100 µg/10 µl, i.t.), or both treatments combined. The antinociceptive effect was evaluated on day 11 for PPF and immediately to (±)-CPP, by assessing the vocalization threshold to mechanical stimulation of the arthritic paw. RESULTS: The data indicate that intrathecal administration of increasing concentrations of (±)-CPP or PPF produced a significant dose-dependent antinociceptive effect with respect to monoarthritic rats receiving saline. The linear regression analysis showed that the dose that produces 30% of maximal effect (ED30) for i.t. (±)-CPP was 3.97 µg, and 1.42 µg for i.t. PPF. The administration of the PPF and (±)-CPP combination in fixed proportions of ED30 produced a dose-dependent antinociceptive effect, showing an interaction of the supraadditive type. CONCLUSIONS: The results suggest that glia inhibitors can synergically potentiate the effect of glutamate blockers for the treatment of chronic inflammatory pain.

Cyclotraxin-B, a new TrkB antagonist, and glial blockade by propentofylline, equally prevent and reverse cold allodynia induced by BDNF or partial infraorbital nerve constriction in mice.

UNLABELLED: Several lines of evidence indicate that brain-derived neurotrophic factor (BDNF) plays a key role as a central pronociceptive modulator of pain, acting through postsynaptic TrkB receptors that trigger intracellular signaling cascades leading to central sensitization. The overall aim of this study was to investigate to what extent BDNF could participate in the generation and maintenance of trigeminal neuropathic pain. The results showed that acute intracisternal administration of nanogram doses of BDNF in naïve mice elicited long-lasting, dose-related, cold allodynic responses to topical application of acetone onto vibrissal pad skin. The systemic administration of cyclotraxin-B (CTX-B), a new TrkB receptor antagonist, or propentofylline, an inhibitor of glial activation, was able to either prevent or reverse the effects of intracisternal BDNF on cold nociception. In addition, the blockade of TrkB receptor by CTX-B inhibited the mechanisms that either initiate or maintain cold allodynia in the ipsilateral vibrissal pad skin after unilateral constriction of the infraorbital nerve. These observations raise the possibility that BDNF is capable on its own of conveying many features of the signaling mechanisms that underlie central sensitization caused by nerve constriction. PERSPECTIVE: Although further studies are necessary to examine in detail the mechanisms underlying the strong anti-allodynic action of CTX-B, this compound may represent an interesting lead for the development of novel therapeutic strategies aimed at preventing and/or suppressing central sensitization associated with neuropathic pain.

Antinociceptive effect of rat D-serine racemase inhibitors, L-serine-O-sulfate, and L-erythro-3-hydroxyaspartate in an arthritic pain model.

N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5'-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 µg/10 µL) and LEHA (100 µg/10 µL) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 µg/10 µL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

Involvement of spinal cord BDNF in the generation and maintenance of chronic neuropathic pain in rats.

Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival and synaptic plasticity of the central and peripheral nervous system. In chronic pain, plastic changes in dorsal horn neurons contribute to a phenomenon of hypersensitivity to pain sensation that is maintained over time, known as central sensitization. This process is accompanied by BDNF overexpression, but the role of BDNF in the generation and maintenance of the hyperalgesic phenomenon is still unclear. The present study was aimed to investigate if exogenous BDNF administered to the rat spinal cord, in addition to trigger pain, participates in the maintenance of the central sensitization process (i.e., pain persistence) and to determine if the pain generated is comparable to that observed in a neuropathic pain model. Results showed that a single intrathecal injection of 0.003 ng of BDNF was able to decrease the nociceptive threshold (Randall-Selitto test) in normal rats, for at least a 42-day period. Furthermore, the hyperalgesia generated was comparable to that observed in rats with a 42-day history of mononeuropathy. Increasing the dose or administering additional doses of BDNF resulted neither in additional effectiveness in reducing the pain threshold nor in the prolongation of the hyperalgesic effect, thus showing that central sensitization induced by BDNF is a dose-independent, all-or-none process. It is concluded that BDNF alone is sufficient for generating a long-lasting neural excitability change in the spinal cord via tyrosine kinase B receptor signaling, similar to that observed in chronic pain models such as neuropathy.

Antihyperalgesic effects of clomipramine and tramadol in a model of posttraumatic trigeminal neuropathic pain in mice.

AIMS: To develop a behavioral model in mice that is capable of mimicking some distinctive symptoms of human posttraumatic trigeminal neuropathic pain such as spontaneous pain, cold allodynia, and chemical÷inflammatory hyperalgesia, and to use this model to investigate the antinociceptive effects of clomipramine and tramadol, two drugs used for the treatment of neuropathic pain. METHODS: A partial tight ligature of the right infraorbital nerve by an intraoral access or a sham procedure was performed. Fourteen days later, mice were subcutaneously injected with saline or drugs and the spontaneous nociceptive behavior, as well as the responses to topical acetone and to formalin or capsaicin injected into the ipsilateral vibrissal pad, were assessed. Data were analyzed by ANOVA. RESULTS: Neuropathic mice exhibited an increased spontaneous rubbing÷scratching of the ipsilateral vibrissal pad, together with enhanced responses to cooling (acetone) and the chemical irritants (formalin, capsaicin). Clomipramine and tramadol produced an antihyperalgesic effect on most of these nociceptive responses, but tramadol was ineffective on capsaicin-induced hyperalgesia. CONCLUSION: Nociceptive responses in this neuropathic pain model in mice exhibited a pattern consistent with the pain described by posttraumatic trigeminal neuropathic patients. The selective antihyperalgesic effect obtained with two commonly used drugs for treating neuropathic pain confirms the validity of this preclinical model.

Effect of interleukin-1beta on spinal cord nociceptive transmission of normal and monoarthritic rats after disruption of glial function.

INTRODUCTION: Cytokines produced by spinal cord glia after peripheral injuries have a relevant role in the maintenance of pain states. Thus, while IL-1beta is overexpressed in the spinal cords of animals submitted to experimental arthritis and other chronic pain models, intrathecal administration of IL-1beta to healthy animals induces hyperalgesia and allodynia and enhances wind-up activity in dorsal horn neurons. METHODS: To investigate the functional contribution of glial cells in the spinal cord nociceptive transmission, the effect of intrathecally administered IL-1beta was studied in both normal and adjuvant-induced arthritic rats with or without glial inhibition. Four weeks after induction of monoarthritis, rats were treated with the glial cell inhibitor propentofylline (10 microg i.t. daily during 10 days) and submitted to a C-fiber-mediated reflex paradigm evoked by single and repetitive (wind-up) electric stimulation. RESULTS: Both the propentofylline treatment and the monoarthritic condition modified the stimulating current required for threshold activation of C reflex responses. Intrathecal IL-1beta increased spinal cord wind-up activity in normal and monoarthritic rats without propentofylline pre-treatment, but resulted in decreased wind-up activity in normal and monoarthritic propentofylline-treated animals. Intrathecal saline did not produce any effect. Thus, glial inactivation reverted into inhibition the excitatory effect of IL-1beta on spinal cord wind-up, irrespective of the normal or monoarthritic condition of rats. CONCLUSIONS: The results suggest that the excitatory effect of nanomolar doses of IL-1beta on spinal wind-up in healthy rats is produced by an unidentified glial mediator, while the inhibitory effects of IL-1beta on wind-up activity in animals with inactivated glia resulted from a direct effect of the cytokine on dorsal horn neurons. The present study failed to demonstrate a differential sensitivity of normal and monoarthritic rats to IL-1beta administration into the spinal cord and to disruption of beta glial function, as both normal and monoarthritic animals changes wind-up activity in the same direction after propentofylline treatment, suggesting that after glial inhibition normal and monoarthritic animals behave similarly relative to the capability of dorsal horn neurons to generate wind-up activity when repeatedly stimulated by C-fibers.

Antinociceptive effect and interaction of uncompetitive and competitive NMDA receptor antagonists upon capsaicin and paw pressure testing in normal and monoarthritic rats.

We assessed whether intrathecal administration of the uncompetitive and competitive NMDA receptor antagonists ketamine and (+/-)CPP, respectively, could produce differential modulation on chemical and mechanical nociception in normal and monoarthritic rats. In addition, the antinociceptive interaction of ketamine and (+/-)CPP on monoarthritic pain was also studied using isobolographic analysis. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the tibio-tarsal joint. Four weeks later, the antinociceptive effect of intrathecal administration of the drugs alone or combined was evaluated by using the intraplantar capsaicin and the paw pressure tests. Ketamine (0.1, 1, 10, 30, 100, 300 and 1000 microg i.t.) and (+/-)CPP (0.125, 2.5, 7.5, 12.5, 25 and 50 microg i.t.) produced significantly greater dose-dependent antinociception in the capsaicin than in the paw pressure test. Irrespective of the nociceptive test employed, both antagonists showed greater antinociceptive activity in monoarthritic than in healthy rats. Combinations produced synergy of a supra-additive nature in the capsaicin test, but only additive antinociception in paw pressure testing. The efficacy of the drugs, alone or combined, is likely to depend on the differential sensitivity of tonic versus phasic pain and/or chemical versus mechanical pain to NMDA antagonists.

Agmatine induces antihyperalgesic effects in diabetic rats and a superadditive interaction with R(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid, a N-methyl-D-aspartate-receptor antagonist.

Agmatine, an endogenous cationic amine resulting from the decarboxylation of L-arginine, produces antihyperalgesic and antiallodynic effects in animal models of chronic neuropathic and inflammatory pain. We examined the effect of agmatine on tactile and thermal allodynia and on mechanical hyperalgesia in streptozocin-induced diabetic rats. To determine its mechanism of action and the potential interest of some of its combinations, the antihyperalgesic effect of agmatine was challenged with alpha(2)-adrenergic imidazoline and opioid-receptor antagonists, and its interaction with the opioid-receptor agonist morphine, the competitive N-methyl-D-aspartate receptor antagonist D-CPP [R(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid], and the nitric-oxide synthase inhibitor L-NAME (L-N(G)-nitro-L-arginine methyl ester) were examined. When intrathecally (i.t.) injected (4.4 to 438 nmol/rat), agmatine was ineffective in normal rats but suppressed tactile allodynia (von Frey hair test), thermal allodynia (tail immersion test), and mechanical hyperalgesia (paw-pressure test) in diabetic rats. This spinal antihyperalgesic effect was suppressed by idazoxan (40 micromol/rat i.t.) but not by yohimbine (40 micromol/rat i.t.) or naloxone (0.69 micromol/rat i.v.). In diabetic rats, an isobolographic analysis showed that combinations of i.t. agmatine with i.v. L-NAME or with i.t. morphine resulted in an additive antihyperalgesic effect, whereas the agmatine/D-CPP i.t. combination was superadditive. In summary, the present findings reveal that spinal agmatine produces antiallodynic and antihyperalgesic effects in diabetic neuropathic pain involving, at least for its antihyperalgesic effect, the imidazoline receptors. Moreover, agmatine combined with D-CPP produces an antinociceptive synergy in experimental neuropathy, opening opportunities in the development of new strategies for pain therapy.

Expression of nitric oxide synthase isoforms in the dorsal horn of monoarthritic rats: effects of competitive and uncompetitive N-methyl-D-aspartate antagonists.

Chronic pain is associated with N-methyl-D-aspartate (NMDA) receptor activation and downstream production of nitric oxide, which has a pivotal role in multisynaptic local circuit nociceptive processing in the spinal cord. The formation of nitric oxide is catalyzed by three major nitric oxide synthase (NOS) isoforms (neuronal, nNOS; inducible, iNOS; endothelial, eNOS), which are increased in the spinal cord of rodents subjected to some tonic and chronic forms of experimental pain. Despite the important role of NOS in spinal cord nociceptive transmission, there have been no studies exploring the effect of NMDA receptor blockade on NOS expression in the dorsal horn during chronic pain. Furthermore, NOS isoforms have not been fully characterized in the dorsal horn of animals subjected to arthritic pain. The aim of this work was therefore to study the expression of nNOS, iNOS and eNOS in the dorsal horns of monoarthritic rats, and the modifications in NOS expression induced by pharmacological blockade of spinal cord NMDA receptors. Monoarthritis was produced by intra-articular injection of complete Freund's adjuvant into the right tibio-tarsal joint. At week 4, monoarthritic rats were given either the competitive NMDA antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or the uncompetitive NMDA antagonist ketamine. After 6 and 24 hours, animals were killed and posterior quadrants of the lumbar spinal cord were dissected. Sample tissues were homogenized and subjected to immunoblotting with anti-nNOS, anti-iNOS or anti-eNOS monoclonal antibodies. The nNOS isoform, but not the iNOS and eNOS isoforms, were detected in the dorsal horns of control rats. Monoarthritis increased the expression of nNOS, iNOS and eNOS in the dorsal horns ipsilateral and contralateral to the inflamed hindpaw. Intrathecal administration of CPP and ketamine reduced nNOS expression in monoarthritic rats but increased the expression of iNOS and eNOS. Results suggest that blockade of spinal cord NMDA receptors produces complex regulatory changes in the expression of NOS isoforms in monoarthritic rats that may be relevant for nitridergic neuronal/glial mechanisms involved in the pathophysiology of monoarthritis and in the pharmacological response to drugs interacting with NMDA receptors.