In clinical situations, most of the patients suffer from inflammation and acute postoperative pain after surgery. Postoperative pain has been emphasized as a very crucial issue in improving the quality of medical care in each medical center. Therefore, management of the postoperative pain is an effective approach to reduce the painful unpleasant feeling, complications, and death rate after surgery. Surgical trauma results in the induction of COX-2, leading to the release of prostaglandins, which sensitize peripheral nociceptors and increase the excitability of spinal neurons, producing pain hypersensitivity in the surrounding uninjured tissue.The purpose of this study is to test the preventive effect of COX-2 inhibitor SC-236 for post-operative pain by rat plantar incision model.Then, we explored whether SC-236 is more effective in reducing the hyperalgesia and inflammation response administered before incision than after incision. Furthermore, we used male Sprague-Dawley rats received plantar incision were used in this study, the rats received subcutaneous injection of SC-236 before or after plantar incision. Behavior teste of mechanical allodynia¡Bthermal hyperalgesia and COX-2 expression level was determined at 4 h and 1, 2, and 3 days after surgery. Mechanical allodynia was measured by mechanical withdrawal threshold that was determined by stimulating with von Frey filaments stimulation. Thermal hyperalgesia was measured by thermal withdrawal thermal tested by radioactive thermal assay. Mechanical allodynia¡Bthermal hyperalgesia and COX-2 expression level were measured at various time points by behavior teste¡Breal-time polymerase chain reaction¡Bwestern blot and immunohistochemistry. The data from pre-incisional injection of SC-236 was compared with that from post-incisional injection of SC-236.The results revealed pre-incisional injection of COX-2 inhibitor significantly inhibited thermal hyperalgesia but not mechanical allodynia then post-incisional injection of COX-2 inhibitor group. Skin of pre-incisional injection of SC-236 show significant decreased mRNA expression of COX-2 at 1 day and 2 day after incision evidenced by real-time polymerase chain reaction. Western blot and immunohistochemistry also show significant decreased protein expression of COX-2 at 4 hours and 1 day after incision. Therefore, pre-incisional administration of SC-236 could prevent the surgical wound induced thermal hyperalgesia and decrease mRNA and protein expression level of cutaneous COX-2 at 4 hours and one day after surgical incision compared to post-incisional administration of SC-236 .

During my research career in Prof. V.J.Hruby's laboratory I worked on two different projects. The first project, which was initiated by the author, was planned to serve the need of our laboratory for a novel method of peptide cyclization. This method was planned to use recent advances in Pd0-catalyzed asymmetric synthesis combined with the structural richness offered by the Baylis-Hillman chemistry which could open new ways to diverse areas of drug design, molecular immunology and chemotherapy. This approach would provide cyclic peptides featuring N-alkylated amino acids that would confer high resistance to degradation by proteases. Because of numerous synthetic problems imposed, this strategy was not of considerable current use in peptide synthesis, especially on solid supports. However, despite a substantial amount of effort invested, this method faced serious drawbacks such as multistep synthesis and side reactions when applied to solid supports. Moreover, recent introduction of microwave technology which has helped to solve a great number of problems has led to a renaissance in the classical lactam and thioester bond cyclizations which overshadowed our quest for a novel methodology. The second project was focused on application of 4-anilidopiperidines for the synthesis of chimeric bioactive peptides. It was an effort towards the development of novel analgesics with reduced toxicity and enhanced potency. This project linked small molecule and multimeric ligand designs that were ongoing in our laboratory at the time. Major accomplishments in this project were made possible by successful resolution of several research challenges. I was able to find a straightforward, convenient and economical approach for the synthesis of novel analogues on a solid support. These developments led to novel compounds which showed substantial increases in their binding affinity relative to corresponding opioid analogues. To illustrate, compounds PET25, 26, 27, 29, 30, 31, and 32 showed high bioactivity and sub-nanomolar binding affinity to opioid receptors. Most of the peptides generated in the second project are still being investigated for their biological activities by our colleagues at the Department of Pharmacology, but the results to date indicate that some highly potent novel compounds have been made.

Fosfolipases A2 miotóxicas (Lys49, enzimaticamente inativa, e Asp49, com atividade) isoladas do veneno de Bothrops asper, induzem hipernocicepção. Assim, avaliamos os mecanismos estruturais, moleculares e mediadores centrais envolvidos neste efeito. A injeção intraplantar das FLA2s acarretou hiperalgesia, enquanto que apenas a FLA2-Asp49 induziu alodinia. A região C-terminal é a responsável pelo efeito da FLA2-Lys49, enquanto que a atividade catalítica da FLA2-Asp49 parece ser responsável pela indução de hipernocicepção. Canais de Ca2+ e Na+ participam deste efeito. Na medula espinhal, receptores NK1 e para CGRP, receptores ionotrópicos para glutamato, NO, IL-1, prostanóides e adenosina participam da hiperalgesia induzida pelas FLA2s. Adicionalmente, receptores metabotrópicos para glutamato e o TNF?, estão envolvidos na hiperalgesia induzida pela FLA2-Asp49. Receptores NK1 e NK2 e para CGRP, receptores para glutamato, TNF? e prostanóides medeiam a alodinia. A ativação de astrócitos e microglia, na medula espinhal, contribui para a gênese do efeito hipernociceptivo. / Phospholipase A2 (Lys49, catalytically-inactive and Asp49, catalytically active), isolated from Bothrops asper snake venom, induce pain. The present studies examined the molecular, structural and central mechanisms involved in hypernociception induced by both PLA2s. These PLA2s induced mechanical hyperalgesia, whereas only PLA2-Lys49 evoked allodynia. The C-terminal region of the PLA2-Lys49 seems to be responsible for hyperalgesia, whereas the enzymatic activity of PLA2-Asp49 contributes to such an effect. Calcium and sodium channels are involved in PLA2s-induced hyperalgesia. In the spinal cord, NK1 and CGRP receptors, glutamate ionotropic receptors, NO, IL-1, prostanoids and adenosine contribute to hyperalgesia caused by PLA2s. Additionally, metabotropic glutamate receptors and TNF are involved in hyperalgesia induced by PLA2-Asp49. NK1, NK2 and CGRP receptors, glutamate receptors, TNF and prostanoids mediate allodynia. Activation of spinal astrocytes and microglia contribute to the generation of hyperalgesia and allodynia induced by both toxins.

Odontalgia Atípica (OA) é uma condição dolorosa orofacial crônica de intensidade moderada a severa, que ocorre nas estruturas dentoalveolares e na mucosa oral. É considerada de difícil diagnóstico por estar associada com a ausência de alterações clínicas e radiográficas perceptíveis. Seus aspectos patofisiológicos sensoriais e de manutenção e perpetuação da dor ainda são mal compreendidos. Os objetivos deste estudo foram: (1) avaliar as alterações somatossensoriais em pacientes com OA através dos testes sensoriais quantitativos (QST); (2) ampliar o conhecimento disponível sobre os mecanismos de modulação da dor através do teste de controle de modulação da dor (CPM); e (3) avaliar as condições psicológicas como ansiedade e depressão, qualidade do sono e qualidade de vida através de questionários auto-aplicáveis. Um total de 50 indivíduos foram incluídos, sendo 25 sujeitos do grupo sintomático com OA (19 mulheres, 58,25 +- 12,17 anos de idade) e 25 sujeitos saudáveis do grupo controle (19 mulheres, 58,92 +- 7,39 anos)(p>0.05). Os QSTs englobaram os testes de Limiar de Detecção Mecânica (MDT), Limiar de Sensibilidade Dolorosa Mecânica (PDT), Teste Mecânico de Alodinia com cotonete (DMA1) e escova dental (DMA2), Testes de Detecção Dolorosa do tipo quente (HPD) e gelado (CPD) e o Teste de Somação Temporal (WUR). O controle de modulação da dor foi feito através do teste CPM e as avaliações psicológicas através do Inventário de Ansiedade e Depressão de Beck, o Índice de Qualidade do Sono de Pittsburg e o Questionário de Qualidade de Vida SF-36. Os QSTs foram repetidos após a aplicação de uma pomada anestésica de Benzocaína 2%. A análise estatística foi feita através dos testes t pareado, teste t e o teste não paramétrico de Mann-Whitney, considerando-se um nível de significância de 5%. Os resultados indicaram que sujeitos com OA apresentam ganho sensorial por meio de estímulos térmicos do tipo quente (HPD) e gelado (CPD) e estímulos mecânicos dinâmicos (DMA1, DMA2 e WUR), e perda sensorial à estímulos mecânicos (MDT, PDT). Ainda, o teste CPM reduziu a intensidade da dor significativamente somente para o grupo controle. A aplicação tópica de anestesia indicou uma redução significativa na intensidade da dor nos indivíduos afetados. Além disso, sujeitos com OA apresentaram sintomas de maior depressão e ansiedade, qualidade do sono ruim e baixa qualidade de vida comparados à pacientes saudáveis. Este estudo enfatizou que alterações somatossensoriais significativas são encontradas em sujeitos com OA, onde envolve uma participação de processos periféricos de sensitização e condução da dor, associado à fenômenos de alodínia e hiperalgesia, o que sugere alterações em nível de sensitização central. O sistema modulátorio de dor mostra-se deficiente e as condições psicológicas estão afetadas em sujeitos com OA. / Atypical Odontalgia (AO) is a chronic orofacial painful condition, which occurs in dentoalveolar structures and oral mucosa. AO is difficult to diagnose because it is associated with the absence of any clinical and radiographic alterations. Repetitive dental procedures are made, with the aim to relief pain. Sensory pathophysiological aspects and pain maintenance and perpetuation are still poorly understood. The aim of this study were: (1) evaluate somatosensory abnormalities in AO patients through quantitative sensory testing (QST), (2) evaluate mechanisms of pain modulation through the controlled pain modulation test (CPM), and (3) assess the psychological features such as anxiety, depression, sleep quality and quality of life through selfreported questionnaires. A total of 50 subjects were included, consisting of 25 subjects with symptomatic AO (19 women, 58.25 +- 12.17 years old) and 25 subjects in the control group (19 women, 58.92 +- 7.39 years old)(p>0.05). QST encompassed Mechanical Detection Threshold (MDT), Pain Detection Threshold (PDT), Dynamical Mechanical Allodynia with a cotton swab (DMA1) and with a toothbrush (DMA2), Cold Pain Detection (CPD), Heat Pain Detection (HPD) and Wind-up Ratio (WUR). Pain modulation was performed by CPM and psychological evaluations through Anxiety Inventory and Beck Depression Index, the Pittsburgh Sleep Quality and Quality of Life Questionnaire SF-36. QSTs were repeated after the administration of an anesthetic cream (2% Benzocaine). Statistical analysis was performed using the \"t\" test, paired t test and nonparametric Mann-Whitney test considering a significance level of 5%. Results indicated that AO subjects showed sensory gain through heat (HPD) and cold (CPD) stimuli and dynamic mechanical stimuli (DMA1, DMA2 and WUR), and sensory loss to mechanical stimuli (MDT, PDT). Moreover, CPM reduced pain intensity significantly only in the control group. A topical anesthesia showed a significant reduction in pain intensity in affected subjects. Furthermore, subjects with AO had symptoms of depression and anxiety, poor sleep quality and poor quality of life compared to healthy individuals. This study emphasized that somatosensory abnormalities are found in subjects with AO, which involves participation of peripheral sensitization associated with allodynia and hyperalgesia, suggesting central sensitization abnormalities. Pain modulation system proves deficient and psychological conditions are affected in subjects with AO.

No description available.

No description available.

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.

No description available.

La migraine constitue un problème de santé publique ayant un impact négatif majeur dans la vie quotidienne des patients. Douze pour cent de la population mondiale souffre de migraine et son coût est évalué à 18 milliards d'euros par an dans la communauté européenne. La fréquence des crises peut augmenter au cours du temps chez certains migraineux, faisant ainsi évoluer une migraine épisodique (0 à 14 jours de crises par mois) en migraine chronique (plus de 15 jours de crises par mois). Ce processus est qualifié de transformation de la migraine. La fréquence des crises semble être un facteur important dans ce processus. Utilisant des approches comportementales, électrophysiologiques, immunohistologiques(expression de la protéine Fos), nous avons identifié une partie des mécanismes neurobiologiques impliqués dans la transformation des céphalées. En effet, en développant un modèle animal de stimulation chimique répétée des méninges nous avons évalué (1) le rôle de l'intensité et de lafréquence sur la sensibilité cutanée (2) la sensibilisation des neurones de la corne dorsale spinale et trigéminale (3) l'efficacité du traitement de fond de référence de la crise, le propranolol (4) l’implication du tronc cérébral (le locus coeruleus (LC) et la substance grise periacqueducale (PAG)) dans la sensibilisation des neurones trigéminaux et spinaux (5) l'implication du LC dans les modifications de la sensibilité cutanée induite par la stimulation chimique des méninges. Notre étude comportementale révèle que la topographie (céphalique et/ou extracéphalique) et la nature (statique ou dynamique) de l'allodynie induite par la stimulation chimique des méninges est sous l'influence de deux facteurs : l'intensité et la fréquence de la stimulation, qui peuvent entraîner sa persistance. Nos études électrophysiologiques et comportementales confirment que l'allodynie cutanée céphalique et extracéphalique est le reflet de la sensibilisation des neurones trigéminaux cervicaux et spinaux, qui peut devenir persistante. Le propranolol, permet de prévenir la modification de la sensibilité cutanée ainsi que l'altération de l'activité des neurones trigéminaux et spinaux. Nous avons également observé la sensibilisation de deux structures du tronc cérébral : la PAG et la PAG qui se traduisent par une altération persistante des CIDN. Cependant le propranolol semble prévenir l'altération de tous ces phénomènes via l'inhibition d'une seule structure le LC et non la PAG. En effet, nos microinjections de propranolol dans le LC permettent de prévenir toutes les modifications induites par la stimulation chimique des méninges. En conclusion ces résultats suggèrent que la fréquence et l'intensité des crises de migraine sont deux facteurs de risque endogène de transformation des céphalées. Un marqueur de cettetransformation est l'allodynie, qui est le reflet clinique de la sensibilisation centrale des neurones de la corne dorsale. Cette sensibilisation centrale est due à l'arrivée directe d'information provenant des méninges mais également à une altération de l'activité neuronale du LC. Nous pouvons également dire que le propranolol constitue un excellent choix dans le traitement de la crise migraineuse étant donné qu'il prévient toutes les modifications induites par la stimulation chimique des méninges et qu'il agit via le LC. / Migraine is a public health problem having a major negative impact on the daily lives of patients. Twelve percent of the world population suffers from migraine and its cost is estimated at $ 18 billion per year in the European Community. The frequency of attacks may increase over time in some migraineurs, thus evolve episodic migraine (0-14 days crises by month) in chronic migraine (more than 15 days of attacks by month). This process is called transformation of migraine. Frequency of headache attacks at baseline appears to be an important risk factor for this process. Using behavioral electrophysiological, and immunohistochemical (expression of Fos protein) approaches, we have identified some of the neurobiological mechanisms involved in the transformation of migraine. Indeed, developing an animal model of repeated chemical stimulation of the dura, we evaluated (1) the role of the intensity and frequency of chemical stimulation (crisis) on skin sensitivity (2) sensitization of second order trigeminal and spinal neurons (3) the effectiveness of font treatment reference of the crisis, propranolol (4) the involvement of the brainstem (locus coeruleus (LC) and periacqueductal grey matter (PAG)) in the sensitization of trigeminal and spinal neurons and (5) the involvement of LC changes in skin sensitivity induced by chemical stimulation of the dura. Our behavioral study reveals that topography (cephalic and / or extracephalic) and nature (static or dynamic) of allodynia induced by chemical stimulation of the dura is under the influence of two factors: intensity and frequency of stimulation, which may lead to its persistence. Our electrophysiological and behavioral studies confirm that the cephalic and extracephalic cutaneous allodynia reflects awareness of trigeminal and spinal neurons, respectively, which may become persistent. Propranolol permits to prevent changes in cutaneous allodynia, and trigeminal and spinal sensitization. We also observed the awareness of two brainstem structures: the PAG and LC which result in a persistent alteration of DNIC. However propranolol appears to prevent the alteration of these phenomena via inhibition of a single structure LC and not the PAG. Indeed, our microinjection of propranolol in the LC can prevent any changes induced by chemical stimulation of the dura. In conclusion these results suggest that the frequency and intensity of migraine attacks are two endogenous risk factors of migraine transformation. A marker of transformation is allodynia, which is the clinical expression of central sensitization of the dorsal horn neurons. This central sensitization is due to the direct arrival of information from the dura but also to an alteration of neuronal activity in LC. Based on these findings, one can suggest that patients with frequent and/or severe migraine attacks take as early as possible a migraine prophylactic treatment. This treatment should prevent the cumulative adverse functional consequences on the central nervous system of the activation of dural nociceptors.

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.