在本實驗室之前的研究中顯示在發炎狀態的大白鼠上以intrathecal (i.t.) 方式給予angiotensin IV (Ang IV) ，Ang IV 是insulin-regulated aminopeptidase (IRAP) inhibitor，可以減少腳掌發炎之大白鼠的疼痛過敏化。然而，這個由Ang IV所產生之效果背後的機制還未被完全釐清。 在這次的實驗中，我們利用由carrageenan引起腳掌腫脹之大鼠進行plantar test來研究Ang IV所產生之抗疼痛過敏化其中可能機制。由於有文獻指出，在發炎狀態的大白鼠上以intrathecal (i.t.) 方式給予oxytocin可產生一劑量相關性的抗疼痛過敏化作用，因此我們推測在大白鼠的脊髓層次上由oxytocin以及Ang IV所個別誘導的抗疼痛過敏化之間可能有關聯性。利用 i.t.單獨給予atosiban (selective oxytocin receptor antagonist) 可以觀察到一個較強烈的疼痛過敏化現象，然而合併給予atosiban，可使Ang IV所產生的抗疼痛過敏化完全被阻斷掉。因此我們推測oxytocin在以intrathecal (i.t.) 方式給予Ang IV而阻斷IRAP的活性，進而產生抗疼痛過敏化作用的過程中是一個主要的IRAP受質。 當我們在carrageenan誘導疼痛過敏化的大白鼠上單獨給予低劑量之oxytocin並沒有產生統計上顯著之抗疼痛過敏作用。然而合併給予oxytocin和Ang IV後，則可以有觀察到Ang IV 能增加並且延長oxytocin所引起的抗疼痛過敏化作用。 就內生性oxytocin來看，電刺激paraventricular hypothalamic nucleus (PVN)已被證實可以增加內生性的oxytocin分泌到脊髓。而這樣的一個神經路徑被發現和疼痛的調節具有很密切的關聯性。從我們的結果中，我們發現在carrageenan誘導疼痛過敏化的大白鼠上，i.t.給予Ang IV可以延長PVN電刺激產生的抗疼痛過敏化作用，由此推測 Ang IV可能可以保護內生性oxytocin不被分解或失去活性。 除了oxytocin之外，IRAP在體外的實驗中被證實可以分解數種具生理活性之peptides 的N-terminal amino acid，其中包括vasopressin, bradykinin 以及 enkephalin等。在這些物質中，bradykinin已被證實在週邊發炎的過程中具有重要的調控效果，若IRAP在週邊組織扮演了一個分解促發炎物質的角色，利用Ang IV也許可以去阻斷IRAP之活性進而增加這些促發炎物質。因此，我們在由carrageenan誘導疼痛過敏化的動物模式上利用腳掌局部注射 Ang IV來探討在發炎部位Ang IV可能的作用。我們猜測Ang IV 可能可以調控不同物質(例如：bradykinin)而在發炎部位產生局部之作用， 但結果顯示在疼痛過敏化以及腫脹程度上，Ang IV並不具有調控週邊組織發炎過程的能力。所以推測Ang IV在週邊發炎位置可能並沒有扮演非常重要的角色。 總而言之，本實驗結果證實了Ang IV以及 oxytocin在發炎大白鼠的脊髓層次上所個別引起之抗疼痛過敏化作用之間具有相關性，其中的機制可能是Ang IV在發炎大白鼠的脊髓層次上藉由抑制IRAP降解oxytocin的活性，進而產生抗疼痛過敏化的效果。 / In our previous study, we showed that intrathecal (i.t.) administration of angiotensin IV (Ang IV), an insulin-regulated aminopeptidase (IRAP) inhibitor, could attenuate hyperalgesia in rats with inflammation. However, the underlying mechanism(s) for this effect of Ang IV was not clarified yet. Using the plantar test in rats with carrageenan-induced paw inflammation, we attempted to investigate the possible mechanism(s) of Ang IV in the present study. Because it has been reported that i.t. administration of oxytocin produced a dose-dependent anti-hyperalgesia effect in rats with inflammation, we speculate that there is a possible correlation between the oxytocin-induced and angiotensin IV-induced anti-hyperalgesia at the spinal level in rats. Using i.t. co-administered atosiban (a selective oxytocin receptor antagonist), the anti-hyperalgesia effect of Ang IV was completely abolished, although a severer hyperalgesia was observed in rats receiving atosiban alone. This indicates that oxytocin could be the major substrate of IRAP responsible for the anti-hyperalgesia caused by intrathecal administration of Ang IV, which blocked the activity of IRAP. Using i.t. administration of oxytocin in rats with carrageenan-induced hyperalgesia, the anti-hyperalgesia effect of oxytocin was potent and significant. When Ang IV was co-administered with the low dose of oxytocin, a significant enhancing effect of Ang IV on anti-hyperalgesia of oxytocin was observed. In view of the endogenous oxytocin, electrical stimulation of the paraventricular hypothalamic nucleus (PVN) has been proved to cause the increase of oxytocin release at the spinal cord. This neural pathway has been found to be highly related to the modulation of pain. In our results, we found that i.t. administration of Ang IV could prolong the anti-hyperalgesia induced by PVN stimulation. This suggests a possible protective effect of Ang IV on endogenous oxytocin degradation/dysfunctioning. In addition to oxytocin, it was well known that IRAP is able to cleave the N-terminal amino acid from several bioactive peptides in vitro, including vasopressin, bradykinin and enkephalin. Among these substrates, bradykinin has been demonstrated to be an important mediator in peripheral inflammation. It is a pro-inflammatory substance that can be enhanced by Ang IV, if the peripheral IRAP plays a role in its degradation. Therefore, we examined the possible local effect of intraplantarly injected Ang IV on the carrageenan-induced hyperalgesia in the same model. However, our results showed no effect of local Ang IV on hyperalgesia and paw edema, indicating that Ang IV may not be able to regulate the peripheral inflammatory process. Overall, the present study verified the possible interaction between the oxytocin-induced and angiotensin IV-induced anti-hyperalgesia at the spinal level in rats with inflammation. It suggests that Ang IV may act though the inhibition of the activity of IRAP to reduce the degradation of oxytocin, thereby lead to anti-hyperalgesia in rats with inflammation.

Animal models are crucial to improve the knowledge of the mechanisms underlying the different pathological processes. These models are also excellent tools to facilitate the research of new targets for the treatment of different diseases and to evaluate the benefit/risk ratio of the potential new treatments. We have focussed this research work in the study of a new potential targets for pain, cognitive and eating disorders using new animal models developed in our laboratory. We first investigated the effects of the interaction between cannabinoids and nicotine on cognitive processes and metabolism using different behavioural models and new experimental devices. In a second part of this work, we investigated new therapeutic targets for neuropathic pain and for this purpose we developed a new behavioural model to improve the study of the therapeutic potential and possible side-effects of novel compounds. / Los modelos animales son cruciales para mejorar el conocimiento sobre los mecanismos que constituyen la base de los diversos procesos patológicos. Estos modelos representan también excelentes herramientas para facilitar la investigación de nuevas dianas para el tratamiento de estas enfermedades y para evaluar el cociente beneficio/riesgo de los nuevos tratamientos potenciales. Este trabajo de investigación se encuentra centrado en el estudio de nuevos dianas terapéuticas para el dolor, los procesos cognitivos y los desórdenes alimentarios utilizando nuevos modelos animales desarrollados en nuestro laboratorio. En primer lugar, hemos investigado los efectos de la interacción entre los cannabinoinoides y la nicotina a nivel los procesos cognitivos y del metabolismo usando diversos modelos comportamentales y nuevos dispositivos experimentales. En una segunda parte de este trabajo, hemos estudiado nuevas dianas terapéuticas para el dolor neuropático y hemos desarrollado para este propósito un nuevo modelo comportamental que permite evaluar el potencial terapéutico y los posibles efectos secundarios de nuevos compuestos.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-05-23T13:58:52Z No. of bitstreams: 1 MaisaOF_DISSERT.pdf: 767871 bytes, checksum: d17c478d78b4175dd0a3f62dd37f0234 (MD5) / Made available in DSpace on 2017-05-23T13:58:52Z (GMT). No. of bitstreams: 1 MaisaOF_DISSERT.pdf: 767871 bytes, checksum: d17c478d78b4175dd0a3f62dd37f0234 (MD5) Previous issue date: 2013-08-29 / This study aimed to evaluate the regenerative and analgesic effects of the aqueous and hydroalcoholic extract of Caesalpinia ferrea pods – daily dosage of 300 mg/kg – on experimental sciatic neuropathy and non-neuropathic peripheral pain. An amount of 82 male Wistar rats was used in the experiment. There were 40 neuropathic rats divided in four groups of ten animals. They were treated with aqueous extract, hydroalcoholic extract, saline solution, and one them was pseudo-operated. The other 42 rats were distributed in six groups of seven animals. Three groups were led to writhing test induced by acetic acid administration, and the other three groups to termal test induced by carrageenan. The groups had different treatments with aqueous and hydroalcoholic extract and saline solution. Spontaneous (motor and pain) and induced (hyperalgesia and flare) behavioural studies were conducted. There were not significant difference between the groups treated or not with the extracts. However, in the third week, the induced behaviors of interdigital and lateral pinch presented significant difference between the groups treated with extracts and the ones treated with saline solution, which suggests analgesic effect for this behaviour. The dosage of 300 mg/kg of aqueous and hydroalcoholic extracts of Caesalpinia ferrea pods in the experimental traumatic sciatic neuropathy may not be used as analgesic. This dosage suggests analgesic effects for the induced behaviour of pinch. It shows they may be tested in studies evaluating axonal regeneration. Histological results suggest a higher axonal integrity in animals treated with the extracts, similar to the sham group / Este estudo avaliou os efeitos regenerativo e analgésico do extrato aquoso (EACF) e hidroalcoólico (EHCF) da vagem do jucá na neuropatia ciática experimental e na dor periférica não neuropática em ratos, em uma dose diária de 300 mg/kg. Foram utilizados 82 ratos machos da linhagem Wistar, distribuídos da seguinte forma: 40 animais neuropáticos divididos em quatro grupos de dez animais, tratados com o EAFC, EHCF, solução salina e um pseudo-operado. Quarenta e dois animais foram distribuídos em seis grupos de sete animais cada, sendo três para realização de testes de contorção abdominal induzida por ácido acético e três para realização de teste térmico induzido por carragenina. Os grupos receberam tratamentos com EAFC, EHCF e solução salina. Estudos comportamentais espontâneos (motricidade e dor) e induzidos (hiperalgesia e reflexos) foram avaliados, não havendo diferença significativa entre os grupos tratados e não tratados, entretanto nos comportamentos induzidos de pinçar interdigital e lateral da região plantar, houve uma diferença significativa entre os grupos tratados com os extratos e tratados com solução salina, , sugerindo um efeito analgésico. A dose de 300mg/kg de EACF e EHCF na neuropatia traumática experimental não pode ser utilizada como analgésico, porém sugerem a existência de efeitos analgésicos para os comportamentos induzidos de pinçar. Os resultados histológicos sugerem uma maior integridade axonal dos animais tratados com os extractos, semelhante ao grupo Sham / 2017-05-23

Orientador: Norberto Perri Moraes / Banca: Glauco Issamu Miyahara / Banca: Antonio Francisco Durighetto Júnior / Banca: César Bataglion / Banca: Ana Cláudia Okamoto / Resumo: Introdução: Síndrome Fibromiálgica (SFM) e Disfunção Temporomandibular (DTM) são enfermidades debilitantes que mostram como principal característica a presença de dor crônica podendo compartilhar de aspectos comuns na fisiopatologia e características clínicas. Tem sido associadas à diminuição do limiar de dor à pressão (LDP) devido a mudanças de processamento da dor em nível de sistema nervoso central sendo que a obtenção do LDP pode ser verificada por algometria de pressão. Objetivos: Determinar a prevalência de SFM em pacientes com DTM e comparar características clínicas e o LDP em músculos mastigatórios e ATMs de pacientes com SFM associada ou não a DTM e controles assintomáticos. Metodologia: A prevalência de SFM foi avaliada em 274 pacientes com DTM. O diagnóstico para DTM seguiu os critérios do RDC/TMD e a SFM os critérios da Academia Americana de Reumatologia. O estudo comparativo incluiu 30 mulheres de mesma faixa etária (Grupo SFM+DTM: 15 mulheres e Grupo DTM: 15 mulheres). Os dados foram analisados através dos testes 2-t ou Mann-Whitney para comparação entre pares, e, qui-quadrado ou teste exato de Fischer para variáveis categóricas. Para avaliar o LDP, foram avaliados 57 indivíduos do sexo feminino de mesma raça e faixa etária, divididos em quatro grupos:. Grupo SFM+DTM: 15 indivíduos com SFM e DTM, Grupo DTM: 15 indivíduos com DTM, Grupo SFM: 12 indivíduos com SFM sem dor orofacial nos últimos três meses e Grupo controle: 15 indivíduos assintomáticos sem história de SFM ou DTM. Avaliou-se por algometria o LDP em músculo masseter, temporal, ATMs e ponto controle no leito ungueal do dedo polegar direito. Os dados obtidos foram analisados pelo teste estatístico de Tukey com nível de significância de 5%. Resultados: Observou-se prevalência de 10,58% de SFM em pacientes com DTM. O Grupo SFM+DTM apresentou idade média de 42,8 anos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Fibromyalgia Syndrome (FMS) and Temporomandibular Disorders (TMD) are debilitating diseases that show as main characteristic the presence of chronic pain which may share common aspects on the physiopathology and clinical characteristics. There have been associated with the decrease in pain at the threshold of the pressure (PPT) due to changes in the processing of the pain at the level of central nervous system and the achievement of the PPT can be verified by the algometry of pressure. Aims: The aim was to determine the prevalence of FMS in patients with TMD and compare the clinical characteristics among these patients and compare the PPT in masticatory muscles of patients with SFM associated or not with TMD and asymptomatic controls. Metodology: The prevalence of FMS was evaluated in 274 patients with TMD. The diagnosis for TMD followed the criteria of RDC/TMD and the SFM followed the criteria of the American Academy of Rheumatology. The comparative study included 30 women of the same age range (Group FMS+TMD: 15 women and TMD Group: 15 women). The data was analyzed by 2-t tests or Mann-Whitney test for comparison between pairs, and chi-square or Fischer Exact test for categorical variants. The study of PPT, included 57 female individuals of the same race and age, divided into four groups: FMS+TMD group: 15 people with FMS and TMD, TMD group: 15 people with TMD, FMS group: 12 people without orofacial pain in the last three months and the control group: 15 asymptomatic individuals with no history of FMS or TMD. The PPT was evaluated by algometry in the masseter muscle, temporal, Temporomandibular Joints and control point in the tumb nail bed. The data was analyzed by the Tukey test with statistical significance level of 5%. Results: The prevalence was 10,58% of FMS in patients with TMD. The FMS+TMD group showed greater intensity of pain... (Complete abstract click electronic access below) / Doutor

A dor é um importante sintoma que sinaliza danos teciduais ou agentes potencialmente prejudiciais ao organismo, evocando respostas sensoriais e motoras de proteção. A dor orofacial apresenta alta prevalência na sociedade atual, sendo esta condição associada a tecidos duros e moles da cabeça, face, pescoço e a estruturas intraorais. Considerando as dores orofaciais de origem músculo-esquelética, destacam-se àquelas causadas pela Disfunção Temporomandibular (DTM). A DTM apresenta etiologia multifatorial, caracterizada por quadros crônicos envolvendo a região cervical, a musculatura mastigatória e a articulação temporomandibular (ATM). Desde que a inflamação das ATMs é considerada a principal causa da dor em pacientes portadores de DTM, a busca por novas opções terapêuticas para esta disfunção envolve estudos desta articulação, abrangendo aspectos fisiológicos, morfológicos e moleculares. Considerando o processo inflamatório e os aspectos moleculares envolvidos no desenvolvimento desta condição, é possível que as enzimas proteolíticas extracelulares, destacando-se as Metaloproteinases da Matriz (MMPs), as quais estão envolvidas na reabsorção de colágeno e de outras macromoléculas, tenham participação ativa neste processo. Em particular, estudos demonstraram que as MMPs estão envolvidas na modulação da dor neuropática, bem como estão presentes no líquido sinovial de portadores de inflamação da ATM. Sendo assim, o objetivo deste trabalho foi avaliar a influência da administração do Adjuvant de Freund (CFA) intraarticular, bilateralmente nas ATMs de ratos, na sensibilidade mecânica e nociceptiva, bem como avaliar a expressão das MMPs, em particular da MMP-2 e MMP-9, no gânglio trigeminal, nas diferentes fases de desenvolvimento da inflamação. Os resultados mostraram que a inflamação das ATMs promoveu alodinia mecânica e hiperalgesia orofacial. Em adição, a administração de doxiciclina (inibidor inespecífico das MMPs) reduziu as alterações na sensibilidade mecânica e nociceptiva. A quantificação das MMPs no gânglio trigeminal demonstrou que o início da inflamação promove aumento da MMP-9 (1 e 3 dias), enquanto que nas fases tardias do processo inflamatório acompanha-se o aumento da expressão da MMP-2 (3, 7 e 10 dias). / Pain is an important symptom that signals tissue damage or potentially harmful agents to the body and evokes sensory and motor protection. The orofacial pain is a type of symptoms that appears in high prevalence in modern society. This painful condition is associated with hard and soft tissues of the head, face, neck and intraoral structures. Considering the pain of musculoskeletal origin, we can highlight those caused by temporomandibular dysfunction (TMD). The TMD has a multifactorial etiology, characterized primarily by chronic conditions involving the neck, the chewing muscles and temporomandibular joint (TMJ). Inflammation of the TMJ is considered the main cause of pain in patients with TMD. Thus, the search for new therapeutic options for this disorder involves studies in the TMJ region encompassing physiological, morphological and molecular aspects. Considering the inflammatory process as the main cause of pain present in TMD, it is extremely important to understand the molecular aspects involved in developing this condition. In this context, extracellular proteolytic enzymes, highlighting the metaloproteniases matrix metalloproteinases (MMPs) play major role in the resorption of collagen and other macromolecules. The proteolytic activity of these MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs), which contribute to the maintenance of metabolic balance and structure of the extracellular matrix. Therefore, the objective of this study was to assess whether the type MMP gelatinases (MMP-2 and MMP-9) of the trigeminal ganglion participate in the development of mechanical allodynia and hyperalgesia in rats orofacial chronic inflammation bilateral TMJ. Our results demonstrated the presence of orofacial hyperalgesia, as well as mechanical allodynia in animals with temporomandibular inflammation induced by CFA and an increase in the expression and activity of gelatinases in the trigeminal ganglion of these animals. Still, there was a decrease in nociceptive orofacial hipersensitivity in animals that received a non-specific inhibitor for MMPs (doxycycline, 30mg/kg/day) for 10 days.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fibromyalgia is a complex syndrome with unknown etiology, characterized by the presence of chronic widespread musculoskeletal pain, having the reduction of the physical/ functional performance as a major comorbidity. Pharmacological treatment of fibromyalgia presents limited effectiveness, making it important to use non-pharmacological therapies, such as physical activity, cognitive behavioral therapy and distracting techniques. The qualification of the environment and the AF has been associated with the treatment of conditions that increase the peripheral and central nociceptive activity. This study proposes the investigation of environmental enrichment (EE), a technique based on physical, sensory and cognitive stimulation, and physical activity (PA) as strategies for fibromyalgia prevention. To this end, 24 male Wistar rats were divided into five intervention groups: 1) EE, 2) PA, 3) EE + PA and 4) control, where they were maintained for 4 weeks. After this time, was diffuse chronic muscle pain induced by two injections of acidic saline in the left gastrocnemius muscle. We evaluated the mechanical paw withdrawal threshold (von Frey electronic), temperature threshold (Hot Plate), neuromuscular activity (Rod Route), ambulation (number of quadrants) through the Open Field in six stages: basal after the 1st, 2nd, 3rd and 4th weeks and 24 hours after induction of diffuse chronic pain model. The animals kept with environmental enrichment and physical activity (EE + PF) showed a significant increase in mechanical and thermal thresholds after the third (P<0.001) and second (P<0002) weeks, respectively, when compared to the other groups. This increase remained at the post-induction assessment, while the other groups showed a significant decrease (P<0.01), suggesting the development of hyperalgesia. In the engine testing, EA + FA group showed a significant increase in neuromuscular activity from the first week compared to the other groups (P<0.01), keeping the increase even after induction, while in the other groups there was a significant decrease in performance (P<0.01), whereas in other groups, there was a significant reduction in weight gain (P<0.01). In the Open Field, a significant increase in the total ambulation in AE + AF group from the 1st week (P<0.01) compared to the other groups. These results suggest an association between environmental enrichment and physical activity as a preventive strategy for chronic musculoskeletal pain and optimization of motor control in a model of diffuse chronic muscle pain. / A fibromialgia é uma síndrome complexa, com etiologia indefinida, caracterizada pela presença de dor musculoesquelética crônica generalizada, tendo, como uma das principais comorbidades, a redução do desempenho físico/funcional. O tratamento farmacológico da fibromialgia apresenta efetividade limitada, tornando importante a utilização de terapias não farmacológicas, destacando-se a atividade física (AF), terapia cognitivo-comportamental e técnicas distratoras. A qualificação do ambiente e a AF tem sido associadas ao tratamento de doenças que aumentam a atividade nociceptiva periférica e central. Assim, o presente estudo propõe a investigação do enriquecimento ambiental (EA), técnica baseada em estimulação física, sensorial e cognitiva, e indução à atividade física (AF) voluntária como recursos para prevenção da fibromialgia em um modelo experimental. Para isso, 24 ratos Wistar machos foram divididos em quatro grupos: (1) EA, (2) AF, (3) EA + AF e (4) controle, mantidos nesses protocolos por 4 semanas. Após este período, foi induzida dor muscular crônica difusa através de dupla injeção de salina ácida no músculo gastrocnêmio esquerdo. Foram avaliados o limiar mecânico de retirada da pata (von Frey eletrônico), limiar térmico (Hot Plate), atividade neuromuscular (Rota Rod), ambulação (nº de quadrantes) através do Campo Aberto, em seis momentos: basal, após a 1ª, 2ª, 3ª e 4ª semanas e 24 horas após indução do modelo de dor crônica difusa. Os animais mantidos com enriquecimento ambiental e atividade física (EA+AF) apresentaram aumento significativo do limiar mecânico e da latência térmica a partir da terceira (P<0,001) e segunda (P<0,002) semanas, respectivamente, quando comparado aos outros grupos. Esse aumento se manteve no momento pós-indução, enquanto nos outros grupos houve uma redução significativa (P<0,02), sugerindo o desenvolvimento de hiperalgesia. Já no teste motor, o grupo AE+AF apresentou aumento significativo na atividade neuromuscular a partir da primeira semana quando comparado aos outros grupos (P<0,01), mantendo esse aumento mesmo após a indução, enquanto nos outros grupos houve uma redução significativa (P<0,02). No Campo Aberto, houve aumento significativo na ambulação total no grupo AE+AF a partir da 1ª semana (P<0,01) em comparação aos demais grupos. Esses resultados sugerem a associação entre o enriquecimento ambiental e a atividade física como uma estratégia preventiva da dor musculoesquelética crônica e otimização do controle motor em um modelo de dor crônica muscular difusa.

Orientador: Cláudia Herrera Tambeli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T19:36:40Z (GMT). No. of bitstreams: 1 Bonet_IvanJoseMagayewski_M.pdf: 8243433 bytes, checksum: a5cdaab90bdcc5bbba66a0e5e4a1293f (MD5) Previous issue date: 2013 / Resumo: O Receptor Potencial Transiente Ankiryn 1 (TRPA1) é um canal não seletivo a cátions importante na fixação do limiar nociceptivos e pertencente à superfamília de canais TRP. É expresso em fibra C-nociceptiva e células não neuronais envolvidas na liberação de mediadores pró-inflamatórios. No presente estudo, investigamos se o TRPA1 contribui para a hiperalgesia induzida pela carragenina em ratos, e se essa contribuição é mediada por mecanismos de inflamação, tais como liberação de citocinas pró-inflamatórias e migração de neutrófilos e/ou sensibilização direta do neurônio aferente primário. Avaliamos a sensibilização do nociceptor induzida pela carragenina utilizando estímulos mecânico (analgesímetro mecânico) e químico (capsaicina), com ou sem bloqueio farmacológico local do receptor TRPA1 pelo seu antagonista seletivo HC 030031. A carragenina induziu hiperalgesia com pico na terceira hora, persistindo até vigésima quarta hora. O bloqueio farmacológico do receptor TRPA1 por co-administração de HC 030031 diminuiu significativamente a hiperalgesia induzida pela carragenina na terceira hora e a pós-administração de HC 030031 (2hrs 55min) reduziu na terceira e na sexta hora. O silenciamento do gene do TRPA1, induzido por um pré-tratamento intratecal com Oligonucleotídeo antisense, preveniu a hiperalgesia induzida pela carragenina após 24 horas além de reduzir significativamente a expressão de TRPA1 em células dos gânglios da raiz dorsal (GRD) (L5-6). O tratamento com carragenina, por sua vez, não alterou a expressão do receptor TRPA1 no GRD, e tampouco afetou os níveis de citocinas e a migração de neutrófilos no tecido periférico (patas). Concluímos que TRPA1 tem papel importante no desenvolvimento e manutenção da hiperalgesia inflamatória induzida pela carragenina por contribuir diretamente na excitabilidade do nociceptor. Baseado nesses achados, sugerimos que o bloqueio de TRPA1 é uma estratégia promissora no desenvolvimento de futuras drogas para o controle e tratamento da dor / Abstract: The Transient Receptor Potential Ankiryn 1 (TRPA1) is a nonselective cation channel, important in setting nociceptive threshold belonging to the superfamily of TRP channels. It is expressed in nociceptive C-fibers and in non-neuronal cells involved in pro-inflammatory mediators release. In this study, we asked whether TRPA1 contributes to carrageenan-induced hyperalgesia in rats, and whether this contribution is mediated by mechanisms in inflammation, such as cytokine release and neutrophil migration and/or by a direct sensitization of the primary afferent nociceptors. We measured the carrageenan-induced nociceptive sensitization using a mechanical (mechanical analgesymeter) and a chemical (capsaicin) stimulus, with or without pharmacological blockade of TRPA1 by its selective antagonist HC 030031. Carrageenan-induced Hiperalgesia has peaked at the third hour and persisted until the twenty-fourth hour. Pharmacological blockade of TRPA1 receptor by co-administration of HC 030031 significantly lowered carrageenan-induced hiperalgesia at the third hour and post-administration (2hrs 55min) decreased at both third and sixth hours. The neuronal TRPA1 gene silencing induced by intrathecal pre-treatment with antisense oligodoexynucleotide completely prevented carrageenan-induced hyperalgesia over 24 hours and significantly reduced TRPA1 expression in the dorsal root ganglia cells (DRG ) (L5-6). However, carrageenan treatment, did not affect the TRPA1 expression on DRG, neither affected the cytokines levels and neutrophil migration in peripheral tissue (paws). We conclude that TRPA1 plays an important role in the development and maintenance of carrageenan-induced inflammatory hyperalgesia by directly contributing to nociceptor excitability. Based on these findings, we suggest that TRPA1 blockade is a promising strategy for the development of future drugs to pain treatment and control / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The association between depression and chronic pain is frequent and bidirectional. The high number of patients with depression and chronic pain who are refractory to currently available drugs makes it important search for new drugs to treat these disease. The bis selenide is an organic selenium compound with activities such as antioxidant, antinociceptive and anti-inflammatory in mice. Thus, this study investigated the effect of bis selenide in models of depression and pain in mice, besides the pharmacological mechanisms involved. Oral administration of bis selenide (5mg/kg, p.o.) reduced the immobility time in tail suspension test (TST) with a maximal effect at 1 hour and it remained up to 6 hours in relation to control group. The effect caused by bis selenide in reducing the immobility time was observed at doses of 0.5, 1 and 5 mg/kg in the TST and the forced swimming test (FST) (article 1). The antidepressant-like activity of bis selenide was reversed by pretreatment with p-chlorophenylalanine ethyl ester (PCPA, an inhibitor of synthesis of serotonin), ketanserin (5-HT2A/2C receptor antagonist), ondansetron (5 -HT3 receptor antagonist) (article 1), L-arginine (an amino acid precursor of nitric oxide (NO)), sildenafil (phosphodiesterase inhibitor type 5, PDE-V), S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor) (article 2), cromakalim (potassium channels - K+- opener), minoxidil (K+ channels opener) and GW 9662 (antagonist of peroxisome proliferator activated receptors gamma (PPARγ) (article 3). However, the antidepressant-like effect of SVBS (1 mg / kg, po) was not prevented by pretreatment with prazosin (α1-receptor antagonist), yohimbine (α2-receptor antagonist), propranolol (β-receptor antagonist), SCH 23390 (D1 receptor antagonist), sulpiride (D2 receptor antagonist) and WAY 100635 (selective antagonist of 5-HT1A) (article 1). Furthermore, when administered in a sub-active dose, the bis selenide (0.1 mg / kg, p.o.) showed a synergistic action with fluoxetine (an selective serotonin inhibitor reuptake), NG-nitro-L-arginine (an inhibitor of the NO synthase (NOS)), 7-nitroindazole (7-NI, a specific inhibitor of NOS neurornal), methylene blue (inhibitor of NOS and guanylate cyclase (GC)), 1H-1,2,4] oxadiazole [4, 3-a] quinoxaline-1-one (ODQ, a specific inhibitor of GC) (article 2), tetraethylammonium (TEA, a non-specific inhibitor of K(+) channels), glibenclamide (an ATP-sensitive K(+) channel inhibitor) charibdotoxin (a large and intermediate conductance calcium-activated K(+) channel inhibitor) and apamin (a small-conductance calcium-activated K(+) channel inhibitor) (article 3). The administration of bis selenide (0.1 5 mg/kg, p.o.) also did not modify the activity of monoamine oxidase A and B (MAO-A and MAO-B) and Na+K+ATPase in mice. The levels of nitrite/nitrate in the brain of mice were reduced by treatment of bis selenide (1 mg/kg). Treatment with bis selenide (1 and 5 mg/kg) decreased the depressive-like behavior and mechanical hypernociception in mice subjected to chronic constriction injury (CCI) (article 4). Moreover, the administration of bis selenide (5 and 10 mg/kg, po) showed a reduction in mechanical hyperalgesia and edema in inflammatory models induced by intraplantar (i.pl.) injection of Complete Freund Adjuvant (CFA), carrageenan (Cg) and prostaglandin E2 (PGE2) (article 5). The mechanical hyperalgesia induced by i.pl. injection of CFA or the brachial plexus avulsion (BPA) was attenuated by chronic administration of bis selenide without causing tolerance during treatment. Concomitant administration of bis selenide (5 and 10 mg/kg) with vincristine for 7 days attenuated the mechanical hyperalgesia from 3 to 28 days. According to this study, it may suggest that the mechanisms responsible for the antidepressant-like effect of bis selenide involve the modulation of serotonin (5-HT2A/C and 5-HT3) and PPARγ receptors and the NO/GMPc/K+ pathway. Furthermore, treatment with bis selenide reduced the depressive-like behavior induced by CCI and the mechanical hyperalgesia in inflammatory (CFA, Cg and PGE2) and neuropathic (BPA and vincristine) models. Thus, this study demonstrated that treatment with bis selenide showed antidepressant-like and anti- hyperalgesic effects in different models in mice. / A associação entre a depressão e a dor crônica é freqüente e bidirecional. O elevado número de pacientes com depressão e dores crônicas que são refratários aos medicamentos disponíveis atualmente torna importante a busca por novas drogas para o tratamento destas doenças. O seleneto vinílico bis substituído (SVBS) é um composto orgânico de selênio com atividades antioxidante, antinociceptiva e antiinflamatória em camundongos. Este estudo teve como objetivo investigar o efeito do SVBS em modelos de depressão e dor em camundongos, além dos mecanismos farmacológicos envolvidos. A administração oral do SVBS (5 mg/kg, per oral, p.o.) reduziu o tempo de imobilidade no teste da suspensão da cauda (TSC) com um efeito máximo em 1 hora e permaneceu significativo em relação ao controle até 6 horas. O efeito causado pelo SVBS em reduzir o tempo de imobilidade foi observado nas doses de 0,5, 1 e 5 mg/kg no TSC e no teste do nado forçado (TNF) (artigo 1). A atividade do tipo antidepressiva do SVBS foi revertida pelo tratamento prévio com p-clorofenilalanina etil éster (PCPA, inibidor da síntese de serotonina (5-HT)), cetanserina (antagonista dos receptores 5-HT2A/2C), ondansetrona (antagonista dos receptores 5-HT3) (artigo 1), L-arginina (aminoácido precursor de óxido nítrico (NO)), sildenafil (inibidor da fosfodiesterase tipo 5, PDE-V), S-nitroso-N-acetil-penicilamina (SNAP, um doador de NO) (artigo 2), cromacalina (abrem os canais de potássio - K+), minoxidil (abrem os canais de K+) e GW 9662 (antagonista dos receptores ativados da proliferação de peroxissomos-gama (PPARγ) (artigo 3). Entretanto, o efeito do tipo antidepressivo do SVBS (1 mg/kg, p.o.) não foi prevenido pelo tratamento prévio com prazosin (antagonista de receptores α1), ioimbina (antagonista de receptores α2), propranolol (antagonista de receptores β), SCH 23390 (antagonista de receptores D1), sulpirida (antagonista de receptores D2) e WAY 100635 (antagonista seletivo de receptores 5-HT1A) (artigo 1). Além disso, quando administrado em uma dose sub-ativa, o SVBS (0,1 mg/kg) apresentou uma ação sinérgica com a fluoxetina (antidepressivo inibidor seletivo da recaptação da serotonina), NG-nitro-L-arginina (inibidor da óxido nítrico sintase (NOS)), 7-nitroindazol (7-NI, inibidor seletivo da NOS neuronal), azul de metileno (inibidor da NOS e da guanilato ciclase (GC)), 1H- 1,2,4]oxadiazolo [4,3-a]quinoxalin-1-ona (ODQ, inibidor seletivo da GC) (artigo 2), tetraetilamônio (TEA, bloqueador dos diferentes tipos de canais de K+, inclusive os dependentes de voltagem), glibenclamida (bloqueador de canais de K+ dependente de ATP), caribdotoxina (bloqueador de canais de K+ de alta condutância ativados por cálcio) e apamina (bloqueador de canais de K+ de baixa condutância ativados por cálcio) (artigo 3). A administração do SVBS (0,1-5 mg/kg, p.o.) também não modificou a atividade das enzimas monoamina oxidase-A e B (MAO-A e MAO-B) e Na+K+ATPase no cérebro dos camundongos. Os níveis de nitrito/nitrato no cérebro foram reduzidos com o tratamento com SVBS (1 mg/kg, p.o.). O tratamento com SVBS (1 e 5 mg/kg) diminui o comportamento do tipo depressivo e a dor nos camundongos submetidos à injúria por constrição crônica (ICC) (artigo 4). Além disso, a administração do composto SVBS (5 e 10 mg/kg, p.o.) demonstrou uma redução na hiperalgesia e no edema induzidos pela administração intraplantar (i.pl.) de Adjuvante Completo de Freund (ACF), carragenina (Cg) e prostaglandina E2 (PGE2) (artigo 5). A hiperalgesia mecânica induzida pela administração i.pl. de ACF ou pela avulsão do plexo braquial (ABP) foi atenuada com a administração crônica do SVBS, sem causar tolerância durante o tratamento. A administração concomitante de SVBS (5 e 10 mg/kg, p.o.) com vincristina durante 7 dias atenuou a hiperalgesia mecânica do 3º ao 28º dia de teste. De acordo com o presente estudo pode-se sugerir que os mecanismos responsáveis pela ação do tipo antidepressiva do SVBS envolvem a modulação dos receptores serotonérgicos (5-HT2A/C e 5-HT3), PPARγ e via do NO/GMPc/K+. Além disso, o tratamento com SVBS reduziu o comportamento depressivo induzido pela ICC e a hiperalgesia mecânica em modelos inflamatórios (ACF, Cg e PGE2) e neuropáticos (ABP e vincristina). Desta forma, o presente estudo demonstrou que o tratamento com SVBS apresentou efeitos do tipo antidepressivo e anti-hiperalgésico em diferentes modelos em camundongos.

Nos últimos anos, os estudos de nosso grupo foram focados na área de dor, avaliando diferentes modelos experimentais de dor aguda, neuropática e muscular. A busca por mecanismos moduladores destes tipos de dores são alvo de grande estudo, uma vez que o fenômeno da dor é peculiar e torna-se de difícil tratamento em muitos casos. Distúrbios (VER) musculoesqueléticos são as principais causas de incapacidade nas pessoas durante seus anos de trabalho. Diversos estudos tem sido realizados lesionando o músculo gastrocnêmio como modelo experimental em diferentes animais para melhor entendimento deste tipo de dor. O nosso objetivo foi observar possíveis alterações histológicas no tecido muscular, avaliar a alteração na sensibilidade nociceptiva e a atividade locomotora dos animais após a indução de dor muscular crônica, bem como observar o envolvimento das células gliais na medula espinal destes animais. Em adição, avaliamos a participação de determinadas citocinas com o intuito de obter um perfil inflamatório em nosso modelo experimental. Nossos resultados demonstraram um quadro de inflamação instalada no tecido muscular de animais com miosite crônica através das analises histológicas realizadas. Os testes comportamentais tanto para hiperalgesia mecânica como térmica e alodinia confirmaram a instalação do quadro álgico uma vez que os animais com miosite apresentaram uma queda em seus limiares nociceptivos em relação aos grupos controle. A atividade locomotora dos animais também se demonstrou comprometida após a indução de miosite. Em relação à participação das células gliais neste modelo, demonstramos que houve um aumento na expressão de GFAP e OX-42, correspondentes à marcação astrócitos e células da microglia na porção lombar da medula espinal dos animais com miosite, quando comparados ao grupo controle. Quanto à participação dos mediadores sistêmicos, observamos um aumento nos níveis de IL-1&#946; e fractalquina (FKN) no sangue dos animais, enquanto o nível de IL-10 permaneceu baixo em relação ao grupo controle. Com nossos achados esperamos colaborar com o aprimoramento de estratégias terapêuticas para tratamento de dores musculares. / In the last years, the studies of our group were focused on the area of pain, evaluating different experimental models of acute, neuropathic and muscular pain. The search for mechanisms modulating types of pain are the subject of great study, since the phenomenon of pain is peculiar and becomes difficult in many cases. Musculoskeletal (VER) disorders are the leading causes of disability in people during their working years. Several studies have been carried out with the model of experimental model in different animals to better understand this type of pain. Our objective was to observe the non-muscular histological changes, to evaluate the nociceptive sensitivity and a locomotor activity of the animals after an induction of chronic muscular pain, as well as to observe the involvement of the glial cells in the spinal cord of the animals. In addition, we evaluated the participation of certain cytokines in order to obtain an inflammatory profile in our experimental model. Our results demonstrated a picture of inflammation installed without muscle tissue of animals with chronic myositis through histological analysis. Behavioral tests for both mechanical and thermal hyperalgesia and allodynia confirmed the onset of pain since animals with myositis showed a decrease in their nociceptive thresholds in relation to the control groups. The locomotor activity of the animals was also shown to be impaired after an induction of myositis. Regarding the participation of glial cells in this model, we demonstrated that there was an increase in the expression of GFAP and OX-42, corresponding to marking astrocytes and microglia cells in the portion of the spinal cord of animals with myositis, when compared to the control group. Regarding the participation of systemic mediators, we observed an increase in the levels of IL-1&#946; and fractalkin (FKN) in the blood of the animals, while the level of IL-10 remained low in relation to the control group. With the findings we hope to collaborate with the improvement of therapeutic strategies for the treatment of muscular pain.

Les neuropeptides endogènes chez l’homme, tels que les enképhalines et endomorphines, ont un potentiel thérapeutique considérable dans le traitement de la douleur. Ils agissent en activant les récepteurs opioïdes qui sont très largement distribués dans le système nerveux central ainsi que dans plusieurs tissus périphériques. Ces neuropeptides présentent, cependant, un certain nombre d’inconvénients qui limitent de manière importante leur efficacité thérapeutique. Tout d’abord, en raison de leur hydrophilie, ils ne passent pas la barrière sang/système nerveux, ce qui limite leur accès aux récepteurs opioïdes. De plus, ils présentent un temps de demi-vie plasmatique relativement court du fait d’une métabolisation rapide. Enfin, pour être efficaces, ces neuropeptides devraient résister à la protéolyse dans le système circulatoire et être suffisamment hydrophobes pour traverser ces barrières hémato-nerveuses.Le but de la thèse a consisté à créer de nouveaux nanomédicaments à base d’endorphines pour vectoriser et combattre la douleur de manière efficace.. Dans ce but, a été établi une liaison chimique covalente, enzymatiquement clivable (ester ou amide), entre le squalène (SQ, un lipide naturel et biocompatible) et le neuropeptide. Ce couplage donne lieu à des prodrogues qui ont la capacité de s'auto-assembler en nanoparticules (NPs) dans l'eau sans l’aide d'un agent tensio-actif. D’une manière générale, cette technologie présente de nombreux avantages tels qu’un taux de charge élevé en principe actif, une protection efficace de celui-ci vis-à-vis de la métabolisation et l’absence de phénomène de « burst release ».Durant ce travail de thèse, nous avons pu montrer pour la première fois que les Leu-enképhalines (LENK) pouvaient devenir efficaces pharmacologiquement une fois couplées au squalène, via une liaison amide (Am), ou via un bras espaceur, tel que le dioxycarbonyl (Diox), ou le diglycolate (Dig). Les prodrogues résultant de ce couplage ont toutes montré des propriétés d’auto-assemblage en milieu aqueux. Cette nanoformulation à base de squalène a permis, d’une part de protéger la LENK de la métabolisation rapide dans le plasma et d’autre part, de lui conférer un effet anti-hyperalgésique significatif dans un modèle de douleur inflammatoire induite chez le rat par la carragénine (test de Hargreaves). Il est important de souligner que cet effet anti-hyperalgésique a duré 3 fois plus longtemps qu’avec la morphine. Un prétraitement avec un antagoniste des récepteurs opioїdes imperméable à la BHE, comme la méthylnaloxone a complètement antagonisé l’effet anti-hyperalgésique des nanoparticules de LENK-SQ, démontrant ainsi que celles-ci agissent via les récepteurs opioïdes périphériques. De plus, l’étude de biodistribution de NPs LENK-SQ fluorescentes a montré une forte accumulation des celles-ci au niveau de la patte œdémateuse, mais aussi dans le foie, la rate et les poumons alors qu’aucun signal n’a pu être détecté au niveau cérébral, ce qui confirme bien l’effet périphérique de ces nanoparticules. Enfin, des études toxicologiques ont montré que malgré l’accumulation des NPs dans le foie, les taux d’aspartate transaminase (AST) et alanine transaminase (ALT) n’ont pas augmenté garantissant ainsi l’innocuité des NPs LENK-SQ après leur injection i.v. Cette étude représente une approche innovante et prometteuse permettant une distribution ciblée du neuropeptide endogène LENK dans les tissus œdémateux pour soulager efficacement la douleur inflammatoire. / Enkephalin is an endogenous pentapeptide producing potent analgesia by activating opioid receptors located on central and peripheral neuronal cell membranes. However, its clinical use has historically been limited due to pharmacokinetic issues, including restricted plasma stability and blood brain barrier impermeability. The aim of this project is to create a new enkephalin-based nanomedicine targeting pain, using biocompatible and biodegradable materials for drug delivery and targeting purposes, such as squalene (squalenoylation nanotechnology). This nanotechnology presents a new concept with numerous advantages in comparison with the conventional nanocarriers, such as high drug loading and absence of “burst release”. Here, we show for the first time, that the rapidly metabolized Leu-enkephalin (LENK) neuropeptide may become pharmacologically efficient owing to its simple conjugation with the squalene (SQ) using three different chemical linkers, i.e., dioxycarbonyl (Diox), diglycolate (Dig), or amide bond (Am). The resulting prodrugs were able to self-assemble in nanoparticles in aqueous media. This new squalene-based nanoformulation prevented rapid plasma degradation of LENK and conferred to the released neuropeptide a significant anti-hyperalgesic effect in a carrageenan-induced paw edema model in rats (Hargreaves test). It should be stressed that this effect lasted 3 times longer than morphine. Pretreatment with brain impermeant opioid receptor antagonist naloxone methiodide (Nal-M) reversed the nanoparticles induced anti-hyperalgesia, indicating that LENK-SQ NPs acted through peripherally located opioid receptors. Moreover, the biodistribution of DiD-fluorescently labeled LENK-SQ NPs showed a strong accumulation of the fluorescence within the inflamed paw as well as in the liver, spleen, and lung, while no signal could be detected in the brain, confirming the peripheral effect of LENK-SQ NPs. Toxicological studies showed that despite nanoparticles accumulation in the liver, the levels of aspartate transaminase (AST) and alanine transaminase (ALT) were not increased after i.v. injection of LENK-SQ NPs, highlighting thus their safety. This study represents a novel drug targeting approach, allowing the specific delivery of LENK neuropeptide into inflamed tissues for pain alleviation.