Inibição da glicogênio sintase cinase 3 como nova abordagem no controle da dor aguda e crônica: análise dos mecanismos de ação

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2013.A glicogênio sintase cinase 3 (GSK3) é uma cinase serina/treonina quefoi primeiramente isolada e purificada como uma enzima capaz defosforilar e inativar a enzima glicogênio sintase. Dentre as diversasfunções reguladas pela GSK3 a inflamação é uma das mais importantes.O presente estudo investigou o efeito do N-(4-metoxibenzil)-N0-(5-nitro-1,3-tiazol-2-il)uréia (AR-A014418), um inibidor específico daGSK3, na nocicepção aguda e crônica e os mecanismos neurobiológicosenvolvidos nesse efeito. O pré-tratamento dos animais com ARA014418(0,01-1 mg/kg, intraperitoneal, i.p., 30 minutos antes)diminuiu a nocicepção aguda induzida pelo ácido acético e ainflamatória (segunda fase) causada pela formalina, sem afetar anocicepção neurogênica (primeira fase) deste teste; e o AR-A014418(0,1-10 µg/sítio) co-injetado intraplantarmente (i.pl.) com a formalinatambém inibiu a segunda fase deste modelo. Além disso, o ARA014418(0,1-100 ng/sítio) injetado intratecalmente (i.t.) foi capaz dediminuir a nocicepção aguda nas duas fases do teste da formalina. A coadministraçãode AR-A014418 intratecalmente (10 ng/sítio) reduziu anocicepção induzida pelo glutamato, N-metil-D-aspartato (NMDA); (±)-1-aminociclopentano-trans-1,3-ácido dicarboxílico (trans-ACPD), fatorde necrose tumoral alfa (TNF-a) e interleucina-1 beta (IL-1ß). Em outrogrupo experimental, o AR-A014418 (0,3 mg/kg, i.p.) também diminuiua nocicepção crônica, caracterizada pela hiperalgesia ao estímulomecânico (filamento de von Frey) e térmico ao frio (placa fria) causadapela ligação parcial do nervo isquiático (PSNL), um modelo de dorneuropática. O efeito antinociceptivo do AR-A014418 foi!!significativamente reduzido pelo pré-tratamento dos animais com PCPA(100 mg/kg, i.p., um inibidor da síntese de serotonina) e AMPT (100mg/ kg, i.p., um inibidor da tirosina hidroxilase), mas não pelaadministração de L-arginina (600 mg/kg, i.p., um precursor do óxidonítrico). Além disso, o AR-A014418 (0,3 mg/kg, i.p.) preveniu oaumento dos níveis das citocinas TNF-a e IL-1ß na medula espinal decamundongos submetidos à PSNL. Finalmente, a administração de ARA014418(0,1-1 mg/kg, i.p.) não afetou a atividade locomotora dosanimais. Coletivamente, estes resultados fornecem evidências de que oAR-A014418, administrado pelas vias sistêmica, periférica e central;diminuiu a nocicepção aguda e crônica em camundongos. O mecanismoda ação antinociceptiva do AR-A014418 está relacionado, direta ouindiretamente, com a inibição dos receptores glutamatérgicosionotrópicos e metabotrópicos e/ou pela inibição de citocinas próinflamatórias(TNF-a e IL-1ß), bem como pela ativação de vias decontrole inibitório descendentes da dor (serotoninérgicas ecatecolaminérgicas). Assim, o presente trabalho demonstra que ainibição da GSK3 pode ser um novo e interessante alvo farmacológicono tratamento da dor aguda e crônica.Abstract : Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase thatwas first isolated and purified as an enzyme capable of phosphorylatingand inactivating the enzyme glycogen synthase. Among the diversefunctions that are regulated by GSK3, inflammation has recentlyemerged as one of the most important. The present study investigatedthe antinociceptive effects of N-(4-methoxybenzyl)-N0 -(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), a specific inhibitor of GSK3 in acuteand chronic nociception and the neurobiological mechanisms involved.A 30-minute pretreatment with AR-A014418 (0.01-1 mg/kg,intraperitoneal, i.p.) inhibited the nociception induced by an i.p.injection of acetic acid and also decreased the late (inflammatory) phaseof formalin-induced licking, without affecting the responses of the first(neurogenic) phase. In a different set of experiments, AR-A014418 (0.1-10 µg/site) coinjected intraplantarly (i.pl.) with formalin inhibited thelate phase of formalin-induced nociception. Furthermore, AR-A014418intrathecal (i.t.) administration (0.1-100 ng/site) inhibited both phases offormalin-induced licking. In addition, AR-A014418 coinjection (i.t.)inhibited the nociception induced by glutamate, NMDA, (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumornecrosis factor-alpha (TNF-a), and interleukin-1beta (IL-1ß). ARA014418also presented an antihyperalgesic effect on the partial ligationof the sciatic nerve (PSNL), a neuropathic pain model. AR-A014418administered i.p. (0.3 mg/kg) inhibited mechanical (von Frey filament)and cold hyperalgesia (cold plate) induced by PSNL. Pre-administrationof PCPA (100 mg/kg, i.p., inhibitor of serotonin synthesis) and AMPT!!(100 mg/ kg, i.p., inhibitor of tyrosine hydroxylase), but not L-arginine(600 mg/kg, i.p., a nitric oxide precursor), significantly reduced themechanical anti-hyperalgesia elicited by AR-A014418 (0.3 mg/kg, i.p.).Furthermore, the administration of AR-A014418 (0,3 mg/kg)significantly prevented the increase of TNF-a and IL-1ß levels. Finally,intraperitoneal administration of AR-A014418 (0.1-1 mg/kg, i.p.) didnot affect locomotor activity in the open-field test. Collectively, theseresults provide convincing evidence that AR-A014418, given byperipheral, systemic, and central routes, produces antinociception inacute and chronic pain models. The AR-A014418-dependentantinociceptive effects were induced by modulation of the glutamatergicsystem through metabotropic and ionotropic receptors and the inhibitionof the proinflammatory cytokines (TNF-a and IL-1ß), as well asincreases in serotonergic and catecholaminergic pathways. The presentstudy suggests that the inhibition of GSK3 may be a novelpharmacological target for the treatment of acute and chronic pain

Exercício físico de alta intensidade induz hiponocicepção visceral em camundongos: evidência para o envolvimento de opióides endógenos e serotonina

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2010Exercício físico é toda atividade física planejada, estruturada e repetitiva que tem por objetivo a melhora ou manutenção da aptidão física. Atualmente sabe-se que a prática de exercícios físicos exerce benefícios tanto para a saúde física, como mental; além disso, ele é capaz de auxiliar no controle de algumas condições dolorosas. Ainda é desconhecido se há uma intensidade específica de exercício necessária para produzir hiponocicepção, bem como os mecanismos envolvidos neste efeito. Para responder a estas questões foi avaliada a influência de 5 dias de natação (30 min/dia, água a 37ºC) sobre o limiar nociceptivo de camundongos Swiss machos no teste das contorções abdominais induzidas pelo ácido acético (0,6%, i.p.). Inicialmente foi avaliada a resposta nociceptiva de dois grupos controles: (1) (não-exercitado) submetidos a 30 segundos de nado/dia e (2) (não-exercitado) submetidos a 30 minutos de permanência na água rasa aquecida, estes grupos apresentaram respostas nociceptivas similares. Nos experimentos subseqüentes foi utilizado somente o grupo controle 1. Os animais exercitados apresentaram inibição de 44±3% do número das contorções abdominais quando comparados ao grupo controle 1 (não-exercitado, 30s/dia). Através do nível de lactato sanguíneo foi demonstrado que o domínio, em relação à intensidade de esforço, foi severo neste protocolo de natação. Em seguida foi avaliado o decurso temporal da hiponocicepção induzida pela natação, a mesma mostrou-se efetiva na diminuição da nocicepção visceral 12h e 24h após o exercício, com inibições de 43±10 e 45±4%, respectivamente. Foi evidenciada a participação do sistema opióide e da glândula adrenal na atividade hiponociceptiva do nado, pois: a naloxona (1mg/kg, i.p.) e a adrenalectomia (retirada bilateral das adrenais) reverteram a atividade hiponociceptiva da natação, respectivamente. Além disso, foi demonstrado que o sistema serotoninérgico também participa no efeito hiponociceptivo do nado, tendo em vista que o pré-tratamento dos animais, por 4 dias consecutivos, com ?-clorofenilalanina metil éster (PCPA, 100 mg/kg, i.p., um inibidor da síntese de serotonina) reverteu a hiponocicepção . A natação não foi capaz de alterar a migração de células inflamatórias ocasionadas pela peritonite induzida pelo ácido acético e não alterou a atividade locomotora dos animais avaliada pelo teste do campo aberto. Estes dados fornecem evidência de que os sistemas: opioidérgico e serotoninérgico participam da atividade hiponociceptiva ocasionada pelo exercício físico em camundongos.Exercise is physical activity that is planned, structured, repetitive, and purposive in the sense that improvement or maintenance of one or more components of physical fitness is an objective. Actually it has been known that physical exercise practice exerts physical and mental health benefits; moreover, it helps in painful conditions control. It is unknown if there is a specific intensity of exercise that produced hyponociception and the mechanisms involved. To address these questions it was evaluated the influence of 5 days swimming (30 min/day, water at 37ºC) above nociceptive threshold of Swiss male mice in the abdominal constriction induced by acetic acid test (0,6%, i.p.). Initially it was evaluated the nociceptive response of two control groups: (1) (non-exercised) submitted to 30 seconds/ day to swimming and (2) (non-exercised) submitted to stay 30 minutes in warmish shallow water/day. These groups had a similar nociceptive response. In the subsequent experiments it was used just control 1 group. The exercised animals had a inhibition of 44±3% in the number of abdominal constrictions when compared to control 1 group (non-exercised 30 seconds/day). Through the blood lactate level it was found that the dominion, in relationship to effort intensity, was severe in this swimming protocol. Following it was measured the time course of the swimming hyponociception, it was effective in the reduction of visceral nociception 12h e 24h after exercise, with inhibition of 43 ± 10 and 45 ± 4%, respectively. It was evidenced the participation of opioid system and adrenal glands in the swimming hyponociceptive activity, because: naloxone (1mg/kg, i.p.) and adrenalectomy (bilateral adrenal glands removal) reverted the swimming hyponociceptive activity, respectively. Besides, it was demonstrated that serotonergic system also participated in the hyponociceptive effect of swimming, the pre-treatment for four consecutive days with ?-chlorophenylalanine methyl ester (PCPA, 100 mg/kg, i.p., an inhibitor of serotonin synthesis) reverted the hyponociception caused by physical exercise. The swimming exercise didn't change the inflammatory cells migration induced by acetic acid peritonitis and did not alter the locomotor activity of the animals evaluated by open field test. These data provide evidence that the opioidergic and serotonergic systems participate in the hyponociceptive activity caused by swimming in mice

Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels

Abstract The present study was undertaken to explore the relative contributions of Cav3.2 T-type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long-lasting mechanical anti-hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mechanisms through which JM produces analgesia

Citral: A monoterpene with prophylactic and therapeutic anti-nociceptive effects in experimental models of acute and chronic pain

Citral (3,7-dimethy1-2,6-octadienal) is an open-chain monoterpenoid present in the essential oils of several medicinal plants. The aim of this work was to evaluate the effects of orally administered citral in experimental models of acute and chronic nociception, inflammation, and gastric ulcers caused by nonsteroidal anti-inflammatory drugs (NSAIDs). Oral treatment with citral significantly inhibited the neurogenic and inflammatory pain responses induced by intra-plantar injection of formalin. Citral also had prophylactic and therapeutic anti-nociceptive effects against mechanical hyperalgesia in plantar incision surgery, chronic regional pain syndrome, and partial ligation of sciatic nerve models, without producing any significant motor dysfunction. In addition, citral markedly attenuated the pain response induced by intra-plantar injection of glutamate and phorbol 12-myristate 13-acetate (PMA, a protein kinase C activator), as well as by intrathecal (i.t.) injection of ionotropic and metabotropic glutamate receptor agonists (N-methyl-D-aspartic acid [NMDA] and 1-amino-1,3-dicarboxycyclopentane [trans-ACPD], respectively), substance P, and cytokine tumour necrosis factor-alpha. However, citral potentiated behaviours indicative of pain caused by i.t, but not intra-plantar, injection of a transient receptor potential vanilloid receptor type 1 (TRPV1) agonist. Finally, the anti-nociceptive action of citral was found to involve significant activation of the 5-HT2A serotonin receptor. The effect of citral was accompanied by a gastro-protective effect against NSAID-induced ulcers. Together, these results show the potential of citral as a new drug for the treatment of pain. (C) 2014 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Additional file 1 of Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels

Additional file 1: Figure S1 (a) Time course of mibefradil mediated reversal of mechanical hypersensitivity in mice with a chronic constriction injury of the sciatic nerve. Statistical analyses were performed by two-way ANOVA followed by Tukey's test. Asterisks denote a significant difference of **P < 0.01 and ***P < 0.001 when compared with the control group (n = 5–7). Hashtags denote P < 0.001 for comparison with the sham-operated group. (b) Mibefradil at 10 mg/kg inhibits pain responses in CCI operated wild type mice, but not in Cav3.2 null mice *P < 0.05 and ***P < 0.001 (n = 5–6)

Potential Nociceptive Role of the Thoracolumbar Fascia: A Scope Review Involving In Vivo and Ex Vivo Studies

Nociceptive innervation of the thoracolumbar fascia (TLF) has been investigated over the past few decades; however, these studies have not been compiled or collectively appraised. The purpose of this scoping review was to assess current knowledge regarding nociceptive innervation of the TLF to better inform future mechanistic and clinical TLF research targeting lower back pain (LBP) treatment. PubMed, ScienceDirect, Cochrane, and Embase databases were searched in January 2021 using relevant descriptors encompassing fascia and pain. Eligible studies satisfied the following: (a) published in English; (b) preclinical and clinical (in vivo and ex vivo) studies; (c) original data; (d) included quantification of at least one TLF nociceptive component. Two-phase screening procedures were conducted by a pair of independent reviewers, after which data were extracted and summarized from eligible studies. The search resulted in 257 articles of which 10 met the inclusion criteria. Studies showed histological evidence of nociceptive nerve fibers terminating in lower back fascia, suggesting a TLF contribution to LBP. Noxious chemical injection or electrical stimulation into fascia resulted in longer pain duration and higher pain intensities than injections into subcutaneous tissue or muscle. Pre-clinical and clinical research provides histological and functional evidence of nociceptive innervation of TLF. Additional knowledge of fascial neurological components could impact LBP treatment

Effects of High-Intensity Swimming on Lung Inflammation and Oxidative Stress in a Murine Model of DEP-Induced Injury.

Studies have reported that exposure to diesel exhaust particles (DEPs) induces lung inflammation and increases oxidative stress, and both effects are susceptible to changes via regular aerobic exercise in rehabilitation programs. However, the effects of exercise on lungs exposed to DEP after the cessation of exercise are not clear. Therefore, the aim of this study was to evaluate the effects of high-intensity swimming on lung inflammation and oxidative stress in mice exposed to DEP concomitantly and after exercise cessation. Male Swiss mice were divided into 4 groups: Control (n = 12), Swimming (30 min/day) (n = 8), DEP (3 mg/mL-10 μL/mouse) (n = 9) and DEP+Swimming (n = 8). The high-intensity swimming was characterized by an increase in blood lactate levels greater than 1 mmoL/L between 10th and 30th minutes of exercise. Twenty-four hours after the final exposure to DEP, the anesthetized mice were euthanized, and we counted the number of total and differential inflammatory cells in the bronchoalveolar fluid (BALF), measured the lung homogenate levels of IL-1β, TNF-α, IL-6, INF-ϫ, IL-10, and IL-1ra using ELISA, and measured the levels of glutathione, non-protein thiols (GSH-t and NPSH) and the antioxidant enzymes catalase and glutathione peroxidase (GPx) in the lung. Swimming sessions decreased the number of total cells (p<0.001), neutrophils and lymphocytes (p<0.001; p<0.05) in the BALF, as well as lung levels of IL-1β (p = 0.002), TNF-α (p = 0.003), IL-6 (p = 0.0001) and IFN-ϫ (p = 0.0001). However, the levels of IL-10 (p = 0.01) and IL-1ra (p = 0.0002) increased in the swimming groups compared with the control groups, as did the CAT lung levels (p = 0.0001). Simultaneously, swimming resulted in an increase in the GSH-t and NPSH lung levels in the DEP group (p = 0.0001 and p<0.002). We concluded that in this experimental model, the high-intensity swimming sessions decreased the lung inflammation and oxidative stress status during DEP-induced lung inflammation in mice

MPP+-lesioned mice : an experimental model of motor, emotional, memory/learning, and striatal neurochemical dysfunctions

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces motor and nonmotor dysfunctions resembling Parkinson’s disease (PD); however, studies investigating the effects of 1-methyl-4-phenylpyridinium (MPP+), an active oxidative product of MPTP, are scarce. This study investigated the behavioral and striatal neurochemical changes (related to oxidative damage, glial markers, and neurotrophic factors) 24 h after intracerebroventricular administration of MPP+ (1.8–18 μg/mouse) in C57BL6 mice. MPP+ administration at high dose (18 μg/mouse) altered motor parameters, since it increased the latency to leave the first quadrant and reduced crossing, rearing, and grooming responses in the open-field test and decreased rotarod latency time. MPP+ administration at low dose (1.8 μg/mouse) caused specific nonmotor dysfunctions as it produced a depressive-like effect in the forced swim test and tail suspension test, loss of motivational and self-care behavior in the splash test, anxiety-like effect in the elevated plus maze test, and short-term memory deficit in the step-down inhibitory avoidance task, without altering ambulation. MPP+ at doses of 1.8–18 μg/mouse increased tyrosine hydroxylase (TH) immunocontent and at 18 μg/mouse increased α-synuclein and decreased parkin immunocontent. The astrocytic calcium-binding protein S100B and glial fibrillary acidic protein (GFAP)/S100B ratio was decreased following MPP+ administration (18 μg/mouse). At this highest dose, MPP+ increased the ionized calcium-binding adapter molecule 1 (Iba-1) immunocontent, suggesting microglial activation. Also, MPP+ at a dose of 18 μg/mouse increased thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels and increased glutathione peroxidase (GPx) and hemeoxygenase-1 (HO-1) immunocontent, suggesting a significant role for oxidative stress in the MPP+-induced striatal damage. MPP+ (18 μg/mouse) also increased striatal fibroblast growth factor 2 (FGF-2) and brain-derived neurotrophic factor (BDNF) levels. Moreover, MPP+ decreased tropomyosin receptor kinase B (TrkB) immunocontent. Finally, MPP+ (1.8–18 μg/mouse) increased serum corticosterone levels and did not alter acetylcholinesterase (AChE) activity in the striatum but increased it in cerebral cortex and hippocampus. Collectively, these results indicate that MPP+ administration at low doses may be used as a model of emotional and memory/learning behavioral deficit related to PD and that MPP+ administration at high dose could be useful for analysis of striatal dysfunctions associated with motor deficits in PD