No description available.

P-glycoprotein (Pgp; ABCB1) is a well known transporter involved in energy-dependent-drug efflux activity. At the brain capillary endothelium, its luminal membrane location is ideal for its ascribed role in the physiological efflux of a wide array of structurally and functionally diverse compounds from the brain. This is a critical issue in regards to the delivery of central nervous system (CNS)-acting therapeutics. Moreover, a dysregulation of Pgp has been implicated in specific CNS disease states, including Alzheimer's disease, epilepsy, and brain cancer where an upregulation of Pgp has been well established as a mediator of multi-drug resistance. Inflammation is a common component in all of these conditions. Previously our laboratory has reported changes in BBB molecular and functional properties during inflammatory pain (Huber et al. 2001). This has led us to investigate the effects of peripheral inflammatory pain on Pgp efflux transport properties at the BBB, in vivo. In the present study we examined the effects of lambda-carrageenan-induced inflammatory pain (i.e. hyperalgesia; CIP) on the molecular and functional properties of Pgp at the BBB. Western blots using enriched fractions of isolated rat brain microvessels revealed that Pgp expression at the BBB was increased by CIP and that this increase occurred predominantly within the membrane region of the cell. Additionally, both in situ brain perfusions and whole body antinociceptive profiling of the Pgp substrate and opioid analgesic, [3H] morphine, indicate that changes in Pgp at the BBB, mediated by peripheral inflammation, can impact brain uptake of morphine. To further elucidate the mechanism(s) behind the rapid upregulation (3 h) of Pgp at this region, we explored regulation of Pgp at the plasma membrane. Our findings show that CIP induces a movement of Pgp within these domains and that Pgp co-localizes with caveolin-1 and clathrin, key structural proteins associated with caveolae and clathrin-pit lipid rafts, respectively. Our data indicate for the first time that peripheral inflammatory pain induces functional and molecular changes in Pgp, a critical efflux transporter, at the BBB in vivo and that these alterations may be mediated in part via a proteolipidic re-organization mechanism.

Inflammatory pain is a debilitating condition that can occur following tissue injury or inflammation and results in touch evoked pain (allodynia), exaggerated pain (hyperalgesia) and spontaneous pain, yet the neural plasticity underlying these symptoms is not fully understood. However, it is known that lamina I neurokinin 1 receptor expressing (NK1R+) spinal cord output neurons are crucial for the manifestation of inflammatory pain. There is also evidence that the afferent input to and the postsynaptic response of these neurons may be altered in inflammatory pain, which could be relevant for inflammatory pain hypersensitivity. Therefore, the aim of this thesis was to study inflammatory pain spinal plasticity mechanisms by investigating the synaptic input to lamina I NK1R+ neurons. In ex vivo spinal cord and dorsal root preparations from the rat, electrophysiological techniques were used to assess inflammation-induced changes in and pharmacological manipulation of the primary afferent drive to lamina I NK1R+ neurons. The excitatory input to lamina I NK1R+ neurons was examined and it was found that inflammation did not alter the relative distribution of the type of primary afferent input received and did not potentiate monosynaptic A δ or monosynaptic C-fibre input, the predominant input to these neurons. Spontaneous excitatory input was significantly elevated in the subset of neurons that received monosynaptic A δ-fibre input only, regardless of inflammation. It has recently been shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby ChemR23 agonists can decrease inflammatory pain hypersensitivity, by a mechanism that involves the attenuation of potentiated spinal cord responses. This study has found that the ChemR23 agonist, chemerin, attenuated capsaicin potentiation of excitatory input to lamina I NK1R+ neurons and significantly reduced monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. However, chemerin was without effect in non-potentiated conditions. In exploring potential inflammatory pain spinal plasticity mechanisms, I have investigated a phenomenon called activity-dependent slowing (ADS), whereby repetitive stimulation of C-fibres at frequencies of 1Hz or above results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. This is proposed to limit nociceptive input to the spinal cord, thus regulating plasticity. Results demonstrate that inflammation significantly attenuated C-fibre ADS in isolated dorsal roots. Furthermore, ADS in monosynaptic C-fibre input to lamina I NK1R+ neurons was significantly reduced in inflammatory pain, which could facilitate nociceptive drive to these key spinal cord output neurons and promote inflammatory pain spinal cord plasticity. In conclusion, the major novel findings of this thesis are firstly, that chemerin can attenuate primary afferent input to lamina I NK1R+ neurons in potentiated conditions, which supports recent studies that suggest ChemR23 is a potential target for the development of new analgesics. Secondly, it was discovered that ADS in monosynaptic C-fibre inputs to lamina I NK1R+ neurons is altered in inflammatory pain, which could be relevant for inflammatory pain spinal plasticity. The findings presented in this thesis could contribute to the development of novel inflammatory pain treatments.

Pain is a debilitating condition that presents a problem of clinical relevance. Common analgesics include opioids and non-steroidal anti-inflammatory drugs (NSIADs). Despite different degrees of effectiveness, a major drawback of these analgesic classes is their side effects. For example, side effects associated with opioids include pruritus, respiratory depression, hyperalgesia, constipation, dependence. In addition, chronic use of NSAIDs can cause gastric ulcers. Delta-9 tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, produces antinociception in various preclinical models of pain. Similarly, many synthetic cannabinoid receptor agonists produce antinociception in preclinical models of pain. However, their psychomimetic effects dampen interest for their therapeutic development. THC and these cannabinoids act upon the endocannabinoid system, which is comprised of the cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptors, endogenous ligands arachidonoylethanolamide (anandamide; AEA), 2-arachidonoyolglycerol (2-AG), and endocannabinoid biosynthetic and catabolic enzymes. In the present study, we evaluated the impact of inhibiting one of the major biosynthetic enzymes of 2-AG, diacylglycerol lipase-b (DAGL-b), and two primary endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL), and fatty acid amide hydrolase (FAAH). MAGL is responsible for degrading 2-AG and FAAH is the principal degradative enzyme for anandamide. We hypothesized that inhibiting these enzymes will produce anti-edematous and anti-allodynic effects in preclinical models of inflammatory pain. In Chapter 2, we tested whether the selective MAGL inhibitor JZL184 and FAAH inhibitor PF-3845 would reduce nociceptive behavior in the carrageenan test. JZL184 and PF-3845 significantly attenuated carrageenan-induced paw edema and mechanical allodynia (a nociceptive response to normally non-noxious stimuli), whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Anti-edematous effects of PF-3845 were mediated through CB2 receptors. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Although the data in the first set of studies demonstrate that inhibition of MAGL or FAAH reduces inflammatory pain, JZL184 and PF-3845 only produced partial effects. In an attempt to increase efficacy, Chapter 3 tested whether combined blockade of FAAH and MAGL would produce enhanced anti-edematous and anti-allodynic effects in the carrageenan model of inflammatory pain. Partial blockade of MAGL, with a low dose of JZL184 (4 mg/kg), and full blockade of FAAH, with a high dose of PF-3845 (10 mg/kg), enhanced the anti-allodynic effects, but no further increases in the anti-edematous effects were found. Importantly, repeated administration of this combination did not result in tolerance. A novel FAAH-MAGL dual inhibitor SA-57, which is far more potent in inhibiting FAAH than MAGL, reversed carrageenan-induced allodynia. Taken together, these findings suggest that dual MAGL and FAAH inhibition represents a promising avenue for the treatment of inflammatory pain. Chapter 4 of this dissertation tested whether inhibition of DAGL-b, a major biosynthetic enzyme of 2-AG, would reverse inflammatory pain. Two DAGL-b inhibitors, KT109, and KT172, which have been previously shown to reduce arachidonic acid, prostaglandins, and TNF-a levels in lipopolysaccharide (LPS)-stimulated murine macrophages, were used to test whether these compounds would elicit antinociceptive effects in the LPS model of inflammatory pain model. Because these drugs also inhibit ABHD6, we assessed KT195, a selective ABHD6 inhibitor that is inactive against DAGL-b. KT109, but not KT172 or KT195, significantly reversed LPS-induced allodynia. Importantly, we found that DAGL-b knockout mice possess an anti-allodynic phenotype, but KT109 did not elicit any further decrease in allodynia in these animals. The anti-allodynic effects of KT-109 did not require cannabinoid receptors. Additionally, the anti-allodynic effects of KT-109 did not undergo tolerance following repeated administration. KT-109 did not produce any gastric hemorrhagic effects when compared to the NSAID diclofenac, which significantly produced gastric hemorrhages. These results suggest that blockade of DAGL-b leads to antinociceptive effects through a cannabinoid receptor independent mechanism of action, with absence of notable side effects. Collectively, the research presented in this dissertation suggests that the endocannabinoid catabolic enzymes MAGL and FAAH, and the endocannabinoid biosynthetic enzyme DAGL-b, represent promising targets to treat inflammatory pain.

La prise en charge de la douleur est un enjeu médical majeur car il existe toujours des douleurs réfractaires aux traitements antalgiques actuels. La détection de la douleur est assurée par les neurones nociceptifs dont l'excitabilité est majoritairement contrôlée par les canaux sodiques dépendants du potentiel (Nav). Parmi eux, le canal Nav1.9 se distingue par son expression restreinte dans les nocicepteurs et par ses caractéristiques électrophysiologiques qui lui confèrent un rôle particulier dans l'électrogenèse de ces neurones. Au cours de ce travail de thèse nous avons caractérisé le rôle du canal Nav1.9 dans trois modèles de douleurs inflammatoires : aigue, persistant et chronique. Nous avons mis en oeuvre un ensemble de techniques d'analyses comportementales, moléculaires et électrophysiologiques, qui nous ont permis de montrer le rôle du canal Nav1.9 dans ces trois modèles de douleur et de révéler plusieurs mécanismes de régulation potentiels.Par la suite, nous nous sommes attachés à décortiquer l'un de ces mécanismes. Nous avons montré que le canal Nav1.9 est présent dans des microdomaines membranaires riches en cholestérol. Nous avons mis en évidence que l'inflammation diminuait la quantité de cholestérol dans les tissus. Ce mécanisme est à l'origine de douleurs dues à l'activation des canaux Nav1.9 et à leur relocalisation en dehors des radeaux lipidiques. Enfin nos expériences montrent que l'application topique de cholestérol peut réduire les douleurs inflammatoires, ouvrant de nouvelles perspectives thérapeutiques. / In mammals, perception of pain is initiated by signaling the occurrence of noxious stimuli through nociceptive neurons located in peripheral sensory ganglia. Nociceptive neurons play a pivotal role in pain perception as they transmit painful information to the central nervous system (CNS). They are largely responsible for the modifications of pain sensation caused by a lesion/inflammation or during the course of chronic diseases like rheumatoid arthritis. Unravelling the precise mechanism of ion channel activation during such pathophysiological conditions is one of the most challenging issues to design new therapeutic pain killer strategies.In this PhD thesis work, we will focus on one particular and promising sodium channel, named Nav1.9. We characterized Nav1.9 channel function in three inflammatory pain models: acute, persistent and chronic, using behavioural, molecular and electrophysiological analysis technics. This work allowed us to point out different putative mechanisms of regulation of this channel.We further decipher the regulation of Nav1.9 by cholesterol lipid in membrane microdomains. We showed that Nav1.9 channel is present in rafts specialized membrane microdomains enriched in cholesterol. We demonstrated that inflammation triggers a decrease in cholesterol level in inflamed territories and that cholesterol deletion induces mechanical allodynia in animals. In addition, we demonstrated that this pain was due to Nav1.9 channel activation and relocalization of this channel out of lipid rafts. Finally our experiments reported that exogenous cholesterol application reduces inflammatory pain. All these results provide a new insight in therapeutic perspectives.

No description available.

Chronic pain is a major healthcare concern that directly affects over one hundred million people in the United States alone. While current treatment options like opioids and NSAIDs are effective, they are with significant drawbacks that prevent long term use. It is important to identify and understand new druggable targets for the treatment of pain. Recent findings have demonstrated substance P functions in the RVM to maintain hypersensitivity to noxious heat stimuli in models of persistent peripheral inflammatory injury in a manner dependent on presynaptic NMDA receptors. What remains unclear is how substance P assumes this pronociceptive role following peripheral inflammatory injury. The experiments detailed in this thesis investigated whether the levels and or release of substance P in the RVM was altered following peripheral inflammatory injury. The effect of peripheral inflammatory injury on levels of substance P in the RVM was tested at several time points. The data show that there were no changes in substance P levels in the ipsilateral or contralateral RVM of CFA injected rats compared to their saline controls at any of the time points tested. To assess whether changes in substance P levels occurred in a subset of neurons within the RVM, computer aided densitometry analysis was used to measure substance P immunoreactivity in sections from the RVM of rats treated with CFA or saline. Substance P immunoreactivity was increased in the ipsilateral RVM of the CFA group compared to the corresponding saline sections at the 4 day, but not the 2 week time point. No other changes were observed. Electron microscopy was used to demonstrate the presence of the NMDA receptor and substance P on the same axon terminals within the RVMs of rats treated with either CFA or saline. This colocalization is significant because it identifies NMDA receptors in position to regulate the release of substance P from axon terminals in the RVM. There were no obvious differences in the degree of colocalization between CFA and saline groups. Functional experiments were devised that tested whether substance P release (basal and evoked) in the RVM was increased following peripheral inflammatory injury, and whether said release was regulated by NMDA receptors. The data show that neither basal nor evoked (potassium or veratridine) release was increased following peripheral inflammatory injury. NMDA was able to facilitate the release of substance P in both the CFA and saline treatment groups, but the facilitation was not different between groups. In the absence of any depolarization stimulus, NMDA was unable to elicit any release of substance P beyond basal values. All told, the data show substance P levels in the RVM are not altered by peripheral inflammatory injury. Additionally, neither basal nor evoked release of substance P is altered by peripheral inflammatory injury. The data provide functional and anatomical evidence for modulation of substance P release by glutamate acting at presynaptic NMDA receptors, but do not support the idea of differential modulation of substance P release following peripheral inflammatory injury.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-30T20:39:29Z No. of bitstreams: 1 Tâmara Nogueira Caracterização da atividade antinociceptiva do extrato metanólico de adiantum latifolium Lam. em modelos experimentais de dor.pdf: 916333 bytes, checksum: bd09f406974cde64dc9bb0917d1d11b6 (MD5) / Made available in DSpace on 2012-08-30T20:39:29Z (GMT). No. of bitstreams: 1 Tâmara Nogueira Caracterização da atividade antinociceptiva do extrato metanólico de adiantum latifolium Lam. em modelos experimentais de dor.pdf: 916333 bytes, checksum: bd09f406974cde64dc9bb0917d1d11b6 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Adiantum, um dos gêneros mais amplamente distribuídos da família Pteridaceae é empregado na medicina popular mundialmente. Neste trabalho, nós investigamos as propriedades antinociceptivas do extrato metanólico de Adiantum latifolium (EMA) em modelos animais de dor inflamatória. As propriedades farmacológicas de EMA foram avaliadas nos testes de contorção, formalina, retirada de cauda e nos modelos de edema de pata induzido por carragenina e edema de orelha induzido pelo ácido aracdônico. A toxicidade aguda de EMA, assim como seu efeito sobre o desempenho motor dos camundongos no teste de rota rod, foram investigados. Além disso, o perfil químico de EMA foi avaliado por cromatografia. A administração oral (100-400 mg/Kg) ou intraperitoneal (1-100 mg/Kg) de EMA produziu uma inibição dose-dependente do número de contorções abdominais induzidas pelo ácido acético em camundongos. Do mesmo modo, o tratamento com EMA (100 mg/Kg/IP) inibiu a hipernocicepção induzida pela formalina tanto na fase inicial quanto na fase tardia. Em contraste, EMA não alterou o limiar de resposta a estímulo térmico no teste de retirada de cauda, indicando ausência de ação central. Confirmando sua atividade antiinflamatória, EMA (100 e 200 mg/Kg/IP) inibiu eventos importantes relacionados à resposta inflamatória induzida pela carragenina ou ácido aracdônico: edema local e aumento nos níveis de interleucina-1β tecidual. Camundongos tratados com EMA (200 mg/Kg) não mostraram alteração no desempenho motor no teste de rota rod, ou sinais de toxicidade (1000 mg/Kg) durante um período de 14 dias. A análise fitoquímica preliminar indicou a presença de terpenos, esteróides, flavonóides e ácidos fenólicos, os quais podem ser responsáveis pelos efeitos antinociceptivo e/ou antiinflamatório de EMA. Os extratos metanólicos de diferentes partes da planta apresentaram atividade antinociceptiva de igual magnitude, sugerindo que o princípio ativo de EMA se distribui por toda a planta. Quando as frações do extrato foram avaliadas, a butanólica e de acetato de etila apresentaram maior eficácia, sendo consideradas as frações mais ativas. Nossos resultados demonstram que Adiantum latifolium apresenta consistente atividade antinociceptiva e antiinflamatória em diferentes modelos experimentais, possivelmente pela inibição da produção e/ou liberação de IL-1β, constituindo bom candidato para o desenvolvimento farmacológico. Palavras- / Adiantum, one of the most widely distributed genera of the Pteridaceae family, is employed in folk medicine worldwide. In the present study, we investigated the antinociceptive effects of the methanolic extract of Adiantum latifolium (MEA) in animal models of inflammatory pain. The pharmacological properties of MEA were evaluated by using writhing, formalin and tail flick tests, carrageenan-induced paw oedema and arachidonic acid-induced ear oedema models. Mice motor performance was evaluated in the rota-rod test and the acute toxicity evaluated over 14 days. In the next experiments series, the active part of Adiantum latifolium, as well as the active fraction of MEA, was evaluated. A phytochemical screening for classes of constituents of MEA was carried out by thin layer chromatography (TLC). Oral (100-400 mg/kg) or intraperitoneal (1-100 mg/kg) administration of MEA produced a dose-related inhibition of acetic acid-induced writhing in mice. Furthermore, treatment with MEA (100 mg/kg/IP) inhibited both the early and late phases of formalin induced hypernociception. In contrast, MEA (100 mg/kg/IP) did not prevent the thermal nociception in the tail flick test. In addition, MEA (100 and 200 mg/kg/IP) inhibited important events related to the inflammatory response induced by carrageenan or arachidonic acid: namely local oedema and increase in tissue interleukin-1β levels. MEA (200 mg/kg/IP) treated mice did not show any motor performance alterations. Over the study duration of 14 days, there were no deaths or toxic signs recorded in the group of mice given 1000 mg/kg of MEA. Phytochemical analysis indicated the presence of terpenes, steroids, flavonoids and phenolic acids, which may be responsible for the MEA antinociceptive and/or antiinflammatory effects. Methanolic extracts from different parts of Adiantum latifolium showed equivalent antinociceptive activity, suggesting that the active principle of EMA is homogeny distributed through the plant. Buthanolic and ethyl acetate fractions were the more active fractions of MEA. Our results demonstrate that Adiantum latifolium presents significant antinociceptive and antiinflammatory activities in different experimental models, possibly through an inhibition of IL-1β production and constitute good candidate for pharmacologic development.

Orientador: Carlos Amílcar Parada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T13:34:40Z (GMT). No. of bitstreams: 1 Prado_FilipeCesardo_M.pdf: 428700 bytes, checksum: 1f8f2df5d5cae548c5b0d1a6a66947f7 (MD5) Previous issue date: 2010 / Resumo: Enquanto a hiperalgesia inflamatória depende da liberação de prostaglandinas e/ou de aminas simpatomiméticas que sensibilizam os neurônios aferentes primários, nosso grupo demonstrou recentemente que o bloqueio do receptor P2X3 no tecido periférico previne a hiperalgesia induzida pela carragenina.. No entanto, o mecanismo pelo qual a ativação dos receptores P2X3 neuronais contribui para a hiperalgesia inflamatória não está completamente estabelecido. O presente estudo verifica se a ativação do receptor P2X3 dos neurônios aferentes primários contribui para a hiperalgesia mecânica induzida pela prostaglandina E2 ou pela dopamine no tecido periférico. A co-administração de A317491 (60 µg / paw), um antagonista seletivo do receptor P2X3, ou o prétratamento com dexametasona (1 mg / mL / kg), preveniu a hiperalgesia mecânica medida 3 horas depois da administraçao de carragenina (300 µg / paw) na pata posterior de ratos. A administração de ??meATP (50 µg /paw) induziu hiperalgesia mecânica 1 hora, mas não 3 horas, depois da sua administração, que foi prevenida pela dexametasona ou pelo A317491. Doses sublimiares de PGE2 (4 ng / paw) ou dopamina (0.4 µg / paw) que não induzem hiperalgesia por si só, induziram hiperalgesia, 3 horas depois, quando administradas logo depois de ??meATP ou carragenina em ratos tratados com dexametasona. Esses estados de hiperalgesia ("priming") revelados pelas doses sublimiares de PGE2 ou dopamine foram prevenidos pelo A317491 ou pelo tratamento com administração intraganglionar (DRG-L5) de ODN antisense, mas não pelo ODN mismatch, contra o receptor P2X3 (40 µg /5µL once a day for 4 days). ODN antisense, mas não o ODN mismatch, reduziu a expressão dos receptores P2X3 no nervo safeno e no DRG-L5. Para verificar se a PKC? media esse estado de hiperalgesia, inibidor de translocação de PKC? (1 µg/paw) foi administrado no tecido periférico 45 minutos antes do ??meATP ou PGE2 (100 ng/paw). O inibidor de PKC? preveniu o estado de hiperalgesia induzido pelo ??meATP ("priming"), mas não a hiperalgesia mecânica induzida pela PGE2 (100 ng/paw). Dessa maneira, os resultados desse estudo sugerem que a hiperalgesia inflamatória depended a ativação dos receptores P2X3 neuronais e da subsequente translocação da PKC? , que aumenta a susceptibilidade dos neurônios aferentes primários (priming) à ação de outros mediadores inflamatórios como a PGE2 e as aminas simpatomiméticas / Abstract: While inflammatory hyperalgesia depends on the release of prostaglandins and/or sympathetic amines that ultimately sensitize the primary afferent neurons, we have recently demonstrated that blockade of P2X3 receptor in the peripheral tissue completely prevents carrageenan-induced hyperalgesia. However, the mechanism by which the activation of neuronal P2X3 receptor contributes to the inflammatory hyperalgesia is not completely clear. The present study verifies whether the activation of P2X3 receptor on primary afferent neurons contributes to the mechanical hiperalgesia induced by prostaglandin E2 or dopamine in the peripheral tissue. Co-administration of A317491(60 µg / paw), a selective P2X3,2/3 receptor antagonist, or pre-treatment with dexamethasone (1 mg / mL / Kg), prevented the mechanical hyperalgesia measured 3 hours after the administration of carrageenan (300 µg / paw) in the rat's hind paw. The administration of ??meATP (50 µg /paw) induced mechanical hiperalgesia 1 hour, but not 3 hours, after its administration, which also was prevented by dexamethasone or A317491. Sub-threshold doses of PGE2 (4 ng / paw) or dopamine (0.4 µg / paw) that do not induce hyperalgesia by themselves, induced maximal hyperalgesia, 3 hours after, when administrated Just following ??meATP or carrageenan in rats treated with dexamethasone. These hyperalgesic states ("priming") revealed by sub-threshold doses of PGE2 or dopamine were prevented by A317491 or treatment with ganglionar administrations (DRG-L5) of ODN antisense, but not ODN mismatch, against P2X3 receptor (40 µg /5µL once a day for 4 days). ODN antisense, but not ODN mismatch reduced the expression of P2X3 receptors in the saphenous nerve and in DRG-L5. To verify whether PKC? mediates this hyperalgesic state, PKC? translocation inhibitor (1 µg/paw) was administrated in peripheral tissue 45 min. before ??meATP or PGE2 (100 ng/paw). PKC? inhibitor inhibited the hyperalgesic state induced by ??meATP ("priming"), but not the mechanical hyperalgesia induced by PGE2 (100 ng/paw). Briefly, the findings of this study suggest that the inflammatory hyperalgesia depends on neuronal activation of P2X3 receptor and the subsequent PKC? translocation, which increases the susceptibility of primary afferent neurons (priming) to others inflammatory mediators such as PGE2 and symphatetic amines / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / nÃo hà / Paclitaxel(TaxolÂ), foi o 1 antineoplÃsico efetivo no tratamento de cÃnceres refratÃrios a quimioterapia convencional. Clinicamente, induz mielossupressÃo e neuropatia perifÃrica sensorial dose-limitante e cumulativa, jà bem documentada na literatura. Menos freqÃentemente os pacientes tratados apresentam mialgias e artralgias. No que diz respeito à dor inflamatÃria, nada foi descrito atà o momento, visando correlacionar o envolvimento das citocinas prÃ-inflamatÃrias, com a gÃnese da hiperalgesia associada ao PCX, jà que a droga induz a expressÃo do gene TNF-alpha. Experimentalmente jà foi demonstrado por vÃrios autores, o papel fundamental do TNF-alpha desencadeando uma cascata de citocinas que ativam os dois componentes da dor inflamatÃria (eicosanÃide e simpÃtico). Dados do nosso laboratÃrio registram que o zymosan (ZY) administrado ip em camundongos, induz a liberaÃÃo dessas citocinas por macrÃfagos residentes no modelo de contorÃÃo abdominal (CA), e que sua injeÃÃo intra-articular em joelhos de ratos produz uma periartrite caracterÃstica tida como sinal de hiperalgesia no modelo de incapacitaÃÃo articular (IA). Com base nestes achados, constituiu-se objetivo do presente trabalho, avaliar o efeito do PCX na modulaÃÃo da resposta nociceptiva induzida pelo ZY em dois modelos animais de dor inflamatÃria. Para tanto, injetou-se via ip PCX (8mg/kg/an) 2h antes do ZY (1mg/cav;ip) em camundongos no teste CA. Ratos foram tratados ip com PCX 4 e 8mg/kg/an) 1 h apÃs da administraÃÃo intra-articular ZY (250mcg/an) no teste IA. Em ambos os testes os animais foram prÃ-tratados via Sc com inibidores de COX-1 e COX-2, bloqueador simpÃtico e inibidores de citocinas. Ficou demonstrado que PCX na dose de 8mg/kg potencializa a hiperalgesia inflamatÃria no modelo de CA, aumentando de maneira significativa (p< 0.001) o n de CA em 183%, em relaÃÃo ao controle. Tal efeito foi inibido de maneira significativa (p<0.001) e dose-dependente pela indometacina (ED50 0,05mg/kg), celecoxib (ED50 13,68mg/kg), atenolol (ED50 0,13mg/kg), talidomida (ED50 23,36mg/kg), pentoxifilina (ED50 8,40mg/kg) e dexametasona (ED50 0,71mg/kg). No teste de IA, esse efeito potencializador foi ratificado pelo aumento significativo do tempo de suspensÃo da pata (p< 0.001) na 3 e 4Âh de artrite em relaÃÃo ao controle, na dose 4mg/kg de PCX. Da mesma forma houve inibiÃÃo significativa (p< 0.001) dessa amplificaÃÃo nos ratos prÃ-tratados. Por fim, o PCX (TaxolÂ) amplifica significativamente a hiperalgesia inflamatÃria induzida pelo ZY no modelo de CA e IA, o que justifica em parte as mialgias e artralgias dos pacientes em uso de PCX e indiretamente sugere a participaÃÃo de citocinas hiperalgÃsicas (TNF-alpha), prostanÃides e mediadores simpÃticos na gÃnese deste efeito hipernociceptivo. / Paclitaxel (TaxolÂ) was the first effective antineoplastic in the management of refractory neoplasias to the conventional chemotherapy. It induces clinically to myelosuppression and sensory peripheral neuropathy boundary and cumulative dose, well documented at literature already. Less often the patients exhibited myalgias and arthralgias. As to concern to the inflammatory pain, there is nothing described do date, aiming to correlate the involvement from pro-inflammatory cytokines with the hyperalgesia genesis associated to PCX since the drug induces the alpha-TNF expression gene. It has already been demonstrated, experimentally by several authors, the essential role of alpha-TNF triggering a set of cytokines which active two components of the inflammatory pain (eicosanoid and sympathetic). Findings of our group showed that zymosan (ZY) administrated intraperitoneal (ip) in mice, induces the release of these cytokines by resident macrophages at the wriggling abdominal model (CA) and that their injection intra-articular rat knees produces a feature periarthritis as a sign of hyperalgesia at articular incapacitation model (IA). Based on these findings, the aim of the present work was to evaluate PCX effect at the modulation of nociceptive response induced by zymosan in two animal models of the inflammatory pain. Then, was injected via ip PCX (8mg/kg/an) before two hours of ZY (1mg/cav;ip) in mice on the CA test. Rats were treated with ip PCX (4,8 mg/kg/an) after one hour ZY intra-articular (250mcg/animal;i-art) for IA test. Both tests, the animals were pre-treated subcutaneous via with COX-1 and COX-2 inhibitors, sympathetic blockade and citokines inhibitors. It was demonstrated that PCX (8mg/kg/an) dose potentiates the inflammatory hyperalgesia at CA model, increasing in 183%, being statistical significant at the level p< 0.001, versus experimental group. Such effect was inhibited at level of significance, p< 0.001 and dependent-dose by indomethacin (ED50 0.05mg/kg), celoxib (ED5013.68mg/kg), atenolol(ED50 0.13mg/kg), talidomide (ED50 23.36mg/kg), penthoxyphylin (ED50 8.40mg/kg) and dexamethasone (ED50 0,71mg/kg). This potential effect of at the IA test was justified at IA model in dose 4mg/kg (p< 0,001) by significant increase of time of suspension the paws through arthritis at third and fourth hours versus experimental group. By same way there was significant inhibition at the level p< 0.001 of this magnification of the pre-treated rats. Latter, PCX (TaxolÂ) magnified significantly the inflammatory hyperalgesia induced by ZY at CA and IA models, justifying one sided myalgias and artralgias of the patients by using PCX, suggesting the involvement of the hyperalgesic citokines - alpha-TNF, prostanoids and sympathetic mediators of the genesis of this hyper-nociceptive effect.