No description available.

Chronic inflammatory and neuropathic pain states are poorly understood, and currently inadequately treated. Clinically, the symptoms of such pain states include allodynia (interpretation of innocuous stimuli as noxious), hyperalgesia (increased sensitivity to noxious stimuli) and spontaneous (non-evoked) pain. Additionally, chronic pain states are often associated with affective disorders such as anxiety and depression which can further reduce the individual’s quality of life. It is highly likely that neuropathic pain could occur in combination with chronic inflammation, for example as a result of post-surgical infection. When such injuries occur in early life, during the continuing development of the nervous system, it is possible that longterm adverse changes in sensory processing may occur. To investigate this, we have developed a rodent model of chronic pain with both a neuropathic and inflammatory component, designed to investigate the consequence of these to injuries coinciding. Furthermore, we are also investigating the effect of gestational stress, which has been shown to alter the stress responsiveness of the offspring and may also affect pain processing. To study the effect that prenatal stress may have on pain processing, we have utilised the rodent resident/intruder paradigm as a model of social stress to determine the outcomes of the combination of these adverse perinatal events. We find that a combined inflammatory and neuropathic injury in the adult rat increases sensitivity to both mechanical and thermal stimuli and also increases spontaneous pain, when compared to inflammation or nerve injury alone. We show that neuropathic pain can be induced in neonatal (P8) rats; however there is little response to inflammation at P8 and a combination of these two injuries does not have the additive effect on sensitivity that occurs in the adult. Upon recovery from neonatal nerve injury, we find that a subsequent noxious challenge (formalin) alters nocifensive behaviour, when compared to the formalin response of naïve (no prior injury) animals, indicating long-lasting changes to nociceptive processing. Interestingly, when nerve injury is carried out in adult animals, nocifensive behaviour in response to formalin is not altered compared to naïve controls. Calcium entry through the NMDA receptor and subsequent CaM Kinase IIα activation has been implicated as a crucial factor in long term potentiation (LTP) and the maintenance of sensitised states. In adult models of chronic pain, which may involve LTP mechanisms, we have shown an increased association of CaM Kinase IIα with NR2A/B spinal immunoprecipitates ipsilateral to injury. Furthermore, a different mechanism may be involved in neonatal pain states, as we have shown that spinal CaM Kinase IIα expression increases with development and is present at very low levels at the time of surgery in our pain models. Additionally, a number of other proteins associated with the NMDA receptor complex are developmentally regulated, and their involvement in the initiation and maintenance of chronic pain is likely to differ between the adult and the neonate. We further show that exposure to prenatal stress does not alter the thresholds to mechanical stimuli in adult or early life pain models, however the combination of prenatal stress and postnatal injury results in an enhanced response to formalin in later life, indicating that programming of stress and/or pain pathways has occurred as a result of these early life events. In addition to the development of a novel model of chronic pain, this study highlights the long-term impact that adverse perinatal events can have on offspring.

No description available.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Artrite à uma doenÃa inflamatÃria que afeta as articulaÃÃes sinoviais. Os sintomas mais comuns sÃo o aumento da sensibilidade à dor nas articulaÃÃes (hiperalgesia ou hipernocicepÃÃo) e edema. Uma alternativa para o tratamento da doenÃa à a inclusÃo de plantas medicinais. O Ãleo extraÃdo dos frutos do pequi (Caryocar coriaceum Wittm), tem ampla aplicabilidade na medicina popular. Ensaios prÃ-clÃnicos demonstraram propriedades terapÃuticas do Ãleo, tais como anti-inflamatÃria, gastroprotetora e cicatrizante. Com base neste contexto, o objetivo do presente estudo foi investigar a atividade antinociceptiva e anti-inflamatÃria do Ãleo de pequi (OPCC) na artrite induzida por zymosan em ratos. O estudo foi aprovado pela ComissÃo de Ãtica em Pesquisa Animal da Universidade Federal do Cearà (n 83/11). A artrite foi induzida por uma injeÃÃo intrarticular, no joelho direito, de zymosan (1mg/50ÂL) e foram avaliados a incapacitaÃÃo articular (hipernocicepÃÃo), edema articular, migraÃÃo de leucÃcitos, liberaÃÃo de citocinas e expressÃo de mediadores inflamatÃrios. Os animais foram prÃ-tratados, por via oral, com OPCC (100, 200 e 400mg/kg) ou veÃculo durante 7 dias consecutivos ou em dose Ãnica, 45 minutos, antes da induÃÃo de artrite. Dexametasona, indometacina ou dipirona foram utilizadas como controles positivos. A incapacitaÃÃo articular foi avaliada durante 4 horas apÃs a injeÃÃo de zymosan. Em seguida os animais foram eutanasiados, procedendo-se a lavagem da cavidade articular com EDTA em PBS para avaliaÃÃo de infiltraÃÃo de leucÃcitos, atividade de mieloperoxidase e dosagem de citocinas. O tecido sinovial foi colhido para anÃlise imunohistoquimica de TNF-&#945; e COX-2. TambÃm foram avaliados o edema (diÃmetro transversal da articulaÃÃo) e o aumento da permeabilidade vascular pelo mÃtodo de extravasamento do Azul de Evans. O efeito antinociceptivo foi avaliado ainda atravÃs do teste de hiperalgesia mecÃnica plantar (Von Frey eletrÃnico) utilizando carraginia e PGE2. Os resultados demonstraram uma diminuiÃÃo significativa (p<0,05) da incapacitaÃÃo articular de todos os grupos tratados com OPCC durante 7 dias, no entanto com a dose Ãnica apenas as doses maiores foram eficazes. Em relaÃÃo à migraÃÃo de leucÃcitos para o lÃquido sinovial todos os grupos tratados com o OPCC (dose Ãnica e doses diÃrias) apresentaram significativa reduÃÃo (p<0,05) do nÃmero de leucÃcitos no lavado articular, acompanhada de uma diminuiÃÃo (p<0,05) na atividade da mieloperoxidase. Observou-se uma diminuiÃÃo (p<0,05) da liberaÃÃo de citocinas (TNF-&#945; e IL-1&#946;) no lÃquido sinovial, assim como expressÃo de TNF&#945; e COX-2 no tecido sinovial. O edema foi inibido (p<0,05) com o tratamento com OPCC em todas as doses administradas diariamente. AlÃm disso, os grupos tratados com 100 e 400mg/kg de OPCC apresentaram uma reduÃÃo significativa (p<0,05) da permeabilidade vascular. Os resultados mostraram ainda que a OPCC reduziu significativamente (p < 0,05) a hipernocicepÃÃo induzida pela carragenina e PGE2. Os dados sugerem que o OPCC exibe efeito anti-inflamatÃrio evidenciado pelo modelo de artrite induzida por zymosan em ratos, que pode ser associada com a inibiÃÃo da produÃÃo de citocinas prÃ-inflamatÃrias (TNF-&#945; e IL-1&#946;). Aparentemente essa aÃÃo pode estar envolvida com a inibiÃÃo da migraÃÃo de neutrÃfilos. Sugerimos ainda que o OPCC previne o desenvolvimento da hipernocicepÃÃo inflamatÃria mecÃnica evocada por carragenina e PGE2 (bloqueio direto da hipernocicepÃÃo) em ratos. / Arthritis is an inflammatory disease that affects synovial joints. The most common symptoms are increased sensitivity to joint pain (hyperalgesia or hypernociception) and edema. An alternative to treating the disease is the inclusion of medicinal plants. The oil extracted from the fruits of Pequi (Caryocar coriaceum Wittm) has wide applicability in popular medicine. Pre-clinical tests have demonstrated the therapeutic properties of the oil, such as anti-inflammatory, gastroprotective and healing. Based on this background, the objective of this study was to investigate the antinociceptive and anti-inflammatory oil Pequi (OPCC) in zymosan-induced arthritis in rats. The study was approved by the Ethics Committee on Animal Research of the Federal University of Cearà (n 83/11). Arthritis was induced by intraarticular injection in right knee of zymosan (1mg/50ÂL) and evaluated the articular incapacitation (hypernociception), joint swelling, leukocyte migration, cytokine release and expression of inflammatory mediators. The animals were pretreated orally with OPCC (100, 200 e 400mg/kg) or carrier for 7 consecutive days either as a single dose 45 minutes before the induction of arthritis. Dexamethasone, indomethacin or dipyrone were used as positive controls. The articular incapacitation was assessed 4 hours after injection of zymosan. Then the animals were euthanized, proceeding to wash the joint cavity EDTA in PBS for evaluation of leukocyte infiltration, myeloperoxidase activity and cytokine. The tissue synovial fluid was harvested for immunohistochemical analysis of TNF-&#945; and COX-2. We also evaluated edema (transverse diameter of the joint) and increased vascular permeability by the method of Evans blue extravasation. The antinociceptive effect was evaluated further by testing plant mechanical hyperalgesia (von Frey electronic). The results showed a significant decrease (p <0.05) joint of disability in all groups treated with OPCC for 7 days, but with only a single dose, higher doses were effective. Compared to the migration of leukocytes into the synovial fluid all groups treated with the OPCC (single dose and daily doses) showed a significant reduction p <0.05) in the number of leukocytes in the BAL joints, accompanied by a decrease (p <0.05) in myeloperoxidase activity. There was a decrease (p <0.05) cytokine release (TNF-&#945; and IL-1&#946;) in the synovial fluid as well as expression of TNF and COX-2 in synovial tissue. The edema was inhibited (p <0.05) treatment with all doses in OPCC. Moreover, the groups treated with 100 and 400mg/kg of OPCC showed a significant reduction (p <0.05) in vascular permeability. The results also showed that the OPCC significantly reduced (p <0.05) hypernociception and PGE2 induced by carrageenan. The data suggest that the OPCC exhibits anti-inflammatory effect evidenced by arthritis model in rats induced by zymosan, which can be associated with the inhibition of the production of proinflammatory cytokines (TNF-&#945; and IL-1&#946;). Apparently this action may be involved in the inhibition of neutrophil migration. We also suggest that the OPCC hypernociception prevents the development of carragenan-evoked inflammatory mechanical and PGE2 (direct blockade of hypernociception) in rats.

No description available.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / This work shows for the first time the antinociceptive effect of the citronellyl acetate (CAT) and aimed to characterize the profile of effect and identify possible antinociceptive mechanisms of the cat, in models of acute nociception in mice. The CAT was testeed standardized animal models of pain using mice Swiss (24-32g). The CAT was administered at doses of 25, 50, 75, 100 or 200 mg/kg, by gavage. It was used in the tests of abdominal writhings by acetic acid; hot plate; formalin test; mechanic sensitivity; inflamatory hipernociception induced by carrageennan; Nociception test induced by capsaicin, cinnamaldeide, menthol, acid saline, PMA, 8-Br-cAMP, bradykinin or glutamate, as well as in behavioural models (open field and rota rod tests) that allowed to exclude the possibility of a muscle relaxant action or to induce false-positive result in the earlier models. The results showed that the CAT have antinociceptive effect in the visceral nociception model induced by acetic acid (in the 100 or 200 mg/kg doses, with ED50 of 74.42 mg/kg), this effect was verified after 30 minutes of aplication and continued until 240 minutes (200 mg/kg). Pretreatment with CAT showed antinociceptive effect in licking model induced by intraplantar formalin application and in the thermal nociception model (hot plate). CAT showed the decreasing of mechanical sencibility using the von Frey hair. In the antinociceptive mechanism investigation, CAT showed effect related with K+ATP channels, TRPV1, TRPM8, ASIC, glutamatergics receptors, bradykinin receptors, PKA and PKC. IN the investigation of neurotramitters pathways involved in antinociceptive effect of CAT, we can suggest the involvement of serotonergics system (5-HT1A, 5-HT2A/C receptors) and muscarinic, dopaminergic and &#945;2-adrenergics receptors. With the results showed, we can conclude the CAT have antinociceptive effect in mice, and as possible mechanism the modulation of intracellular mediators PKC and/or PKA, relacted with moleculars mechanisms of K+ATP channels, TRPV1, TRPM8, ASIC, glutamatergics receptors, bradykinin receptors, serotonergics system (5-HT1, 5-HT2A/C receptors) and muscarinic, dopaminergic and &#945;2-adrenergics receptors. / Esse trabalho, atà onde se sabe, mostra pela primeira vez o efeito antinociceptivo do acetato de citronelila (CAT) e teve como objetivo caracterizar o perfil do efeito e identificar possÃveis mecanismos antinociceptivos do CAT, em modelos de nocicepÃÃo aguda em camundongos. O CAT foi testado em modelos animais padronizados de dor utilizando camundongos Swiss (24-32g). O CAT foi administrado nas doses de 25, 50, 75, 100 ou 200 mg/kg, por via oral. Foram utilizados os testes de contorÃÃes abdominais induzidas por Ãcido acÃtico; placa quente; teste da formalina; nocicepÃÃo mecÃnica, hipernocicepÃÃo inflamatÃria induzida pela carragenina; teste da nocicepÃÃo induzida por capsaicina, cinamaldeÃdo, mentol, salina Ãcida, PMA, 8-Br-cAMP, bradicinina ou glutamato, bem como em modelos comportamentais (testes do campo aberto e rota Rod) que permitiram excluir a possibilidade de uma atividade relaxante muscular ou induzir resultados falso-positivos nos modelos anteriores. Os resultados mostraram que o CAT possui efeito antinociceptivo no modelo de nocicepÃÃo visceral induzida por Ãcido acÃtico (nas doses de 100 ou 200 mg/kg, com DE50 de 74,42 mg/kg), esse efeito foi verificado apÃs 30 minutos da aplicaÃÃo e persistiu por atà 240 minutos (200 mg/kg). O prÃ-tratamento com o CAT mostrou efeito antinociceptivo no modelo de lambedura induzida pela aplicaÃÃo intraplantar de formalina e no modelo de nocicepÃÃo tÃrmica (placa quente). O CAT mostrou diminuiÃÃo da sensibilidade mecÃnica utilizando o filamento de von Frey. Na investigaÃÃo do mecanismo antinociceptivo, o CAT mostrou efeito relacionado com os canais K+ATP, TRPV1, TRPM8, ASIC, receptores glutamatÃrgicos, receptores de bradicinina, PKA e PKC. Na investigaÃÃo das vias de neurotransmissÃo envolvidas no efeito antinociceptivo do CAT, podemos sugerir o envolvimento dos receptores &#945;2-adrenÃrgicos, sistema serotonÃrgico (receptores 5-HT1A, 5-HT2A/C), receptores muscarÃnicos e dopaminÃrgicos. Com os resultados mostrados, podemos concluir que o CAT possui efeito antinociceptivo em camundongos, e como possÃveis mecanismos a modulaÃÃo de mediadores intracelulares PKA e/ou PKC, relacionados com os mecanismos moleculares dos canais K+ATP, TRPV1, TRPM8, ASIC, receptores glutamatÃrgico, receptores de bradicinina, receptores &#945;2-adrenÃrgico, sistema serotonÃrgico (receptores 5-HT1, 5-HT2A/C), receptores muscarÃnicos e dopaminÃrgicos.

No description available.

The adverse interaction between smoking and chronic pain has been known for decades. A variety of chronic pain conditions – ranging from headache to low back pain to fibromyalgia – markedly exacerbate smoking prevalence and intensity in packs per day among multiple patient populations. In patients seeking pain treatment, the prevalence of smoking approaches 50%, compared to less than 20% in the general population. Perhaps not surprisingly, the relationship is bidirectional: not only does persistent pain increase rates and intensity of smoking, but smoking also appears to exacerbate both the intensity and associated impairment of chronic pain. In fact, smoking appears to place individuals at risk for developing a chronic pain condition and may also facilitate the transition from acute to chronic pain. The growing body of literature documenting these associations has led to the proposition of a positive feedback loop: individuals smoke in part to cope with their pain, but smoking actually worsens the pain. Despite the strong evidence for the existence of this adverse interaction, the mechanisms responsible for it remain poorly understood. A number of preclinical and clinical studies have documented that nicotinic acetylcholine receptor (nAChR) agonists, e.g., nicotine, have analgesic efficacy in the acute pain setting, such as that produced experimentally in the research laboratory or experienced by patients postoperatively. In contrast, the role of nAChR activation in modulating chronic pain is less well characterized. The experiments described in this thesis determine whether persistent pain diminishes the antinociceptive (analgesic) efficacy of an α4β2 nAChR agonist in the rostral ventromedial medulla (RVM), a key brainstem pain modulatory nucleus, and subsequently begin to elucidate the mechanisms by which persistent pain elicits this plasticity. The complete Freund’s adjuvant (CFA) model of chronic pain was employed to test the hypothesis that persistent inflammatory injury diminishes the antinociceptive efficacy of the selective and potent α4β2 nAChR agonist epibatidine in key brainstem pain modulatory nuclei. Paw withdrawal latency to a noxious heat stimulus was used to evaluate the anti-hyperalgesic and antinociceptive effects of epibatidine microinjected in the RVM or periaqueductal gray (PAG) of male rats. The effects of epibatidine were assessed both in uninjured animals and in animals at different times after intraplantar CFA injection. Interestingly, pretreatment with an α4β2-selective antagonist demonstrated that the antinociceptive effects of epibatidine in naïve rats were mediated by α4β2 nAChRs in the RVM but not in the PAG. While the antinociceptive effects of epibatidine in the RVM were abolished after two weeks of inflammatory pain, the anti-hyperalgesic effects remained unchanged. Surprisingly, epibatidine no longer appeared to be acting primarily at α4β2 nAChRs as early as four hours after injury. Persistent inflammation did not alter the anti-hyperalgesic or antinociceptive effects of epibatidine in the PAG. Radioligand binding studies were conducted to test the most parsimonious hypothesis that a global reduction in α4β2 nAChR number or binding affinity during persistent injury was in part responsible for the decreased efficacy of epibatidine in the RVM after intraplantar CFA. Saturation binding using [3H]-epibatidine in membrane homogenates prepared from RVM and PAG tissue revealed no differences in receptors between saline- and CFA-treated rats at any time after injury, suggesting that a whole-nucleus reduction in nAChRs could not explain the observed behavioral phenomena. To query functional changes with greater resolution, whole-cell patch clamp electrophysiology was employed to begin assessing the consequences of nAChR activation by nicotine at the level of the neuron. Initial studies performed in the locus coeruleus demonstrated that all neurons responded to nicotine with an inward current that desensitized with continued exposure to the drug. Neurons in the RVM exhibited significantly more heterogeneity in their response to nicotine: desensitizing inward currents were seen in some; sustained outward currents in others; inward currents followed by outward currents in a third population; and still others had no response to nicotine exposure. The sustained outward currents persisted in the presence of the sodium channel blocker tetrodotoxin, were not blocked by an α4β2 nAChR-selective antagonist, and appeared to be mediated by G protein-coupled receptors and potassium channels. Taken together, the present results demonstrate that persistent inflammatory injury produces adaptive changes in nicotinic signaling in the RVM such that the antinociceptive effects of epibatidine activation are abolished in a time-dependent manner. These changes cannot be attributed to a whole-nucleus reduction in α4β2 nAChRs. However, nicotinic signaling in the RVM is complex, and small alterations in the pre- or postsynaptic actions of nicotine may have significant ramifications for the overall nociceptive sensitivity of an animal. The data presented here suggest that plasticity in nicotinic signaling within the bulbospinal pain modulatory pathways may in part explain the adverse interaction between smoking and chronic pain observed in humans.

<p>Dendrite shape is a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their functioning depends on these morphologies, remain unclear. Here, we demonstrate a dendrite-specific requirement for the microtubule severing protein Katanin p60-like 1 (Kat-60L1) in regulating the elaborate branch morphology and nocifensive functions of Drosophila melanogaster larval class IV dendritic arborization (da) neuron dendrites. Through genetic loss of function analysis we show that loss of kat-60L1 reduced dendrite branching and process length, particularly during a period of normally extensive growth. This morphological defect was paralleled by a reduction in nocifensive responsiveness mediated by these neurons, indicating a tight correlation between neuronal function and the full extent of the dendritic arbor. To understand the mechanism underlying Kat-60L1's effects, we used in vivo imaging of the microtubule plus-end binding protein EB1, and found fewer polymerizing microtubules within mutant dendrites. Kat-60L1 thus promotes microtubule growth within class IV dendrites to establish the full arbor complexity and nocifensive functions of these neurons. </p><p>Although reduction of the related microtubule severing protein Spastin also compromised class IV dendrite arborization and nocifensive responses, microtubule polymerization in dendrites was unchanged in spastin mutants, and behavioral defects arose from generally compromised neuronal excitation. Kat-60L1 and Spastin thus function in distinct neuronal compartments to establish the complex dendritic morphology and sensory functions of class IV da neurons via distinct mechanisms of microtubule regulation. Whereas Spastin regulates stable microtubules affecting both pre- and post-synaptic compartments of these neurons, Kat-60L1 function is required specifically in dendrites to promote their complex arborization through the addition of growing microtubule numbers. Double mutant analysis demonstrated that Kat-60L1 and Spastin function antagonistically to promote dendritic aborization, likely involving other molecular players involved in regulating the microtubule cytoskeleton. Lastly, we identified Mi-2 as a transcriptional regulator of both kat-60L1 and spastin and show a genetic interaction between mi-2 and kat-60L1 in the class IV dendritic arbor, demonstrating that Mi-2 antagonizes Kat-60L1 function, possibly through the parallel upregulation of spastin. These data support a key role for the differential utilization of microtubule severing in generating distinct neuronal morphologies and subsequent function.</p> / Dissertation

The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.