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Orientador: Antônio Augusto Fasolo Quevedo Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação Resumo: A dor é um dos sintomas mais prevalentes na maioria das afecções clínicas, ao ponto de ser considerado por alguns autores como o quinto sinal vital. Apesar de ser largamente estudada, ainda carece de instrumentos para a sua avaliação de forma objetiva. Enquanto isso, a estimulação elétrica dos tecidos vem sendo largamente utilizada para realização de procedimentos clínicos nas mais variadas áreas, desde a estimulação de tecidos musculares até na analgesia e na recuperação dos tecidos. Esse trabalho visou desenvolver um estimulador elétrico transcutâneo com o objetivo de estimular as fibras nervosas A? e C, responsáveis por transmitir os impulsos sensitivos nociceptivos, e assim mensurar a sensibilidade dolorosa dos indivíduos de maneira objetiva. O aparelho desenvolvido gera uma onda de estimulação com subida e descida abruptas, sendo possível ao operador ajustar a largura do pulso de estimulação, a frequência e a amplitude do estímulo. Estes parâmetros são exibidos em uma tela LCD no dispositivo e podem ser ajustados por botões dispostos abaixo desta tela. O estimulador também possui um espelho de corrente como gerador da onda de estimulação. Essa arquitetura permite que o dispositivo mantenha uma corrente de saída constante, mesmo submetido às impedâncias variáveis da pele íntegra. O dispositivo foi testado em voluntários saudáveis. Foi aplicado a esses voluntários um estímulo de intensidade crescente e ao perceberem o estímulo como doloroso o estímulo era interrompido e os valores de intensidade anotados. O estimulador se mostrou um método eficaz para produzir sensação dolorosa Abstract: Pain is one of the most common symptons in all deseases and considered to be the fifth vital sign for some authors. Although widely studied, it still have few instruments to be evaluated ob-jectively. On the other land, tissue electrical stimulation has been widely used in clinical aplications on most varied areas, like muscular stimulation, pain control and tissue healing, This work deve-loped a transcutaneous electrical stimulator device with the purpose of sensitize A? e C nerve fi-bers, responsible to transmit the nociceptive impulses, and with it mensure subject’s pain sensiti-vity in a objective way. The developed device generates stimulation with abrupt climb and fall, were is possible to the operator to adjust the pulse width, amplitude and frequency. These parameters are displayed in an LCD screen in the device. There are adjustable in bottons below the screen. There is a current mirror in the stimulator that generates the stimulation wave. This architecture allows the device to keep a constant output current even with variable skin impedances. The device was tested in healthy subjects. A growing intensity stimulus was applied to this volunteers, and when they realized a painful sensation, the test was interrupted and the intensity values noted. The stimulator proved to be a effective method to produce painful sensation Mestrado Engenharia Biomédica Mestre em Engenharia Elétrica

Orientador: Antônio Augusto Fasolo Quevedo Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação Resumo: INTRODUÇÃO: A dor é um fenômeno multidimensional com componentes sensitivos, emocio-nais e cognitivos, ligada ou não a um dano orgânico. Mensurar o limiar de dor, de forma padroni-zada, desempenha um papel fundamental para o adequado tratamento da dor. A algometria de pressão mensura a resposta dos nociceptores à pressão, porém apresenta limitações quanto à varia-bilidade dos avaliadores, dificultando o acompanhamento da evolução terapêutica. Um método de algometria elétrica é proposto, utilizando um dispositivo que pode reduzir as limitações causa-das pelo método de pressão e facilitar a prática clínica. OBJETIVOS: Avaliar o limiar de dor por meio de algometria elétrica e a variabilidade interavali-adores; comparar o limiar de dor da algometria elétrica e de pressão por um mesmo avaliador; e avaliar se os limiares de dor por ambos os métodos apresentam diferenças entre os gêneros. METODOLOGIA: Foi realizado um estudo de corte transversal com 60 indivíduos saudáveis do gênero feminino e masculino de 20 a 40 anos, sendo: grupo 1, masculino e grupo 2, feminino, com 30 indivíduos em cada grupo. As avaliações dos limiares de dor foram realizadas com algo-metria elétrica e algometria por pressão. Foi desenvolvido um dispositivo de algometria elétrica em outro trabalho do grupo de pesquisa, e foi realizado um estudo piloto para avaliar os melhores parâmetros para as avaliações. Para avaliar as aplicações clínicas, o dispositivo foi submetido aos estudos de confiabilidade dos avaliadores e, após, foi realizada a etapa de validação clínica do método de algometria elétrica. RESULTADOS E DISCUSSÃO: No estudo de confiabilidade, os resultados mostraram exce-lente confiabilidade intra e interavaliadores para ambos os métodos de algometria. Na etapa de validação clínica com 60 sujeitos, os resultados mostraram que a algometria por pressão pode pre-ver os limiares de dor da algometria elétrica no grupo feminino, quando aplicadas por um mesmo avaliador. Em relação a variabilidade interavaliadores, o algômetro elétrico não mostrou variações significativas em ambos os grupos, mostrando que o método independe do avaliador. Para a com-paração intergrupos, os limiares de dor à pressão não apresentaram diferenças entre os gêneros, conforme já descrito em estudos anteriores. Já os limiares de dor por corrente elétrica obtidos por dois avaliadores mostraram que o grupo masculino apresentou o limiar de dor superior ao do grupo feminino. Nas análises do nível de desconforto, por meio da Escala Visual Analógica da dor (EVA), a algometria elétrica e a algometria por pressão promoveram níveis de desconforto distintos em ambos os gêneros, porém, a algometria elétrica realizada por dois avaliadores promo-veu níveis de desconforto similares dentro de cada gênero e na comparação entre os gêneros, o que confirma a não dependência dos avaliadores para a execução do método e no desconforto gerado pela técnica. CONCLUSÃO: A algometria elétrica mensura o limiar de dor com redução dos fatores limitan-tes da algometria por pressão, em relação à necessidade de treinamento intensivo e variabilidade interavaliadores. Entretanto, a algometria elétrica apresenta diferenças entre os gêneros, porém com níveis de desconforto similares Abstract: INTRODUCTION: Pain is a multidimensional phenomenon with sensitive, emotional and cogni-tive components, linked or not to organic damage. Measuring the pain threshold, in a standardized way, plays a fundamental role for the adequate treatment of pain. Pressure algometry measures the response of nociceptors to pressure, but it has limitations regarding the variability of the evalu-ators, making it difficult to monitor the therapeutic evolution. An electrical algometry method is proposed, using a device that can reduce the limitations caused by the pressure method and facili-tate clinical practice. OBJECTIVES: assess the pain threshold by means of electrical algometry and the inter-rater variability; compare the pain threshold of electrical algometry and pressure by the same evaluator; and assess whether pain thresholds for both methods differ between genders. METHODOLOGY: A cross-sectional study was carried out with 60 healthy female and male in-dividuals aged 20 to 40 years, being: group 1, male and group 2, female, with 30 individuals in each group. Assessments of pain thresholds were performed with electrical algometry and pressure algometry. An electrical algometry device was developed by the research group and a pilot study was carried out to evaluate the best parameters for the evaluations. In order to assess clinical ap-plications, the device was subjected to the reliability studies of the evaluators and, afterwards, the clinical validation step of the electrical algometry method was carried out. RESULTS AND DISCUSSION: In the reliability study, the results showed excellent intra- and inter-rater reliability for both algometry methods. In the clinical validation stage with 60 subjects, the results show that pressure algometry can predict the pain thresholds of electrical algometry in the female group, when applied by the same evaluator. Regarding inter-rater variability, the elec-tric algometer did not show significant variations in both groups, showing that the method is inde-pendent of the evaluator. For intergroup comparison, pressure pain thresholds did not differ be-tween genders, as described in previous studies. The electric current pain thresholds obtained by two evaluators, however, showed that the male group had a pain threshold higher than that of the female group. In the analysis of the level of discomfort, using the Visual Analog Pain Scale (VAS), electrical algometry and pressure algometry promoted different levels of discomfort in both gen-ders, however, the electrical algometry performed by two evaluators promoted similar levels dis-comfort within each gender and in the comparison between genders, which confirms the non-dependence of the evaluators for the execution of the method and the discomfort generated by the technique. CONCLUSION: Electrical algometry measures the pain threshold while reducing limitations of pressure algometry, in relation to the need for intensive training and inter-rater variability. How-ever, electrical algometry has differences between genders, but with similar levels of discomfort Mestrado Engenharia Biomédica Mestra em Engenharia Elétrica

Both TRPA1 and purinergic P2X receptors have been proposed as potential targets for the treatment of visceral pain. We found that the intracolonic administration of a low dose mustard oil (0.5%), a well-known TRPA1 agonist, produced nociceptive responses and abdominal wall referred mechanical hyperalgesia, without inducing apparent tissue damage. Both nociceptive responses and referred hyperalgesia were abolished by the ablation of TRPV1-expressing neurons (and the consequent ablation of TRPA1+ nociceptors) by resiniferatoxin (RTX) treatment, and by the TRPA1 antagonist AP18. However, a higher dose of mustard oil (2.5%) damaged the colonic epithelium and induced pERK activation in the spinal cord, and these processes were clearly independent of TRPV1-expressing neurons ablated by RTX. This higher dose of mustard oil induced nociceptive responses and referred mechanical hyperalgesia which were insensitive or only slightly sensitive to resiniferatoxin or AP18, but were markedly reduced by the P2X antagonist TNP-ATP, which is known to inhibit nociceptive actions induced by ATP released from injured tissues. In conclusion, whereas a low dose of intracolonic mustard oil induces visceral pain in a manner fully dependent on TRPA1 actions, when a high dose of this chemical irritant is used, visceral pain becomes mostly independent of TRPA1 activation but clearly enhanced by ATP purportedly released by the damaged colonic epithelium. Therefore, TRPA1 inhibition is not sufficient to substantially decrease visceral pain during tissue injury, whereas purinergic antagonism appears to be a more effective strategy., Spanish State Research Agency under MINECO PID2019-108691RB-I00, Junta de Andalucia CTS-109

BACKGROUND: Pharmacokinetic (PK)/pharmacodynamic (PD) modelling offers new insights to design protocols for sedation and analgesia in standing horses.OBJECTIVES: To evaluate the parameters and interactions between detomidine and methadone when given alone or combined in standing horses.STUDY DESIGN: Randomised, placebo-controlled, blinded, crossover.METHODS: Eight adult healthy horses were given six treatments intravenously: saline (SAL); detomidine (5 μg/kg bwt; DET); methadone (0.2 mg/kg bwt; MET) alone or combined with detomidine (2.5 [MLD], 5 [MMD] or 10 [MHD] μg/kg bwt). Venous blood samples were obtained at predetermined times between 0 and 360 min after drug administration. Plasma detomidine and methadone were measured using a single, liquid/liquid extraction technique by liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-MS/MS). Sequential PK/PD modelling compared rival models, with and without PK and PD interaction between drugs, to fit the PD data including height of the head above the ground (HHAG), a visual analogue scale for sedation (VAS), electrical (ET), thermal (TT) and mechanical (MT) nociceptive thresholds and gastrointestinal motility (GIM) [1].RESULTS: Two and three compartment models best described the PK of detomidine and methadone, respectively. Detomidine decreased its own clearance as well as the clearance of methadone. The interaction of methadone on the effect of detomidine revealed an infra-additive (partial antagonism) effect for HHAG (α = -1.33), VAS (α = -0.98) and GIM (α = -1.05), a positive potentiation for ET (pot = 0.0041) and TT (pot = 0.133) and a synergistic to additive effect for MT (α = 0.78).MAIN LIMITATIONS: This is a small experimental study.CONCLUSIONS: Different PK/PD interactions were demonstrated for each PD parameter and could be modelled in vivo. The modelling of our data will allow us to simulate and predict the effect of constant rate infusions of both drugs for future investigations.

Actas del V Congreso de Señalización Celular, SECUAH 2020. 16-18 de marzo, 2020. Universidad de Alcalá. Alcalá de Henares, Madrid. España., La insensibilidad congénita al dolor es una condición congénita rara, siendo el tipo IV de la enfermedad, que además presenta anhidrosis, el más frecuente entre la población y para el cual actualmente no existe tratamiento y los estudios al respecto son puramente teóricos. En este trabajo teórico proponemos el estudio molecular de la enfermedad para permitir el desarrollo de terapias encaminadas a solventar el problema. Para ello, estudiamos el gen NTRK1, gen codificante del receptor tirosina quinasa TrkA, que actúa como regulador de la diferenciación de neuronas sensoriales y que en este caso, se encuentra mutado. El estudio de esta mutación y sus consecuencias se realizó mediante ensayos de secuenciación y genotipado de individuos, así como también se propuso una terapia génica con CRISPR/Cas9 en modelos in vivo y diversos ensayos in vitro mediante western-blot y citometría de flujo. Nuestros resultados muestran la posibilidad de diseñar un diagnóstico de portadores y de aplicar la terapia génica con el fin de revertir la mutación del gen y conseguir el correcto funcionamiento de las neuronas. La existencia de conocimiento teórico sobre la enfermedad y la posibilidad del diseño de terapias posibles para ella constituyen un paso previo para investigar las causas y consecuencias funcionales de la misma

Una amputación es un suceso que afecta la calidad de vida, la cual compromete diferentes tareas diarias, también priva al individuo de una fuente de información que le permite interactuar con el mundo físico; por ende, una solución prometedora es la rehabilitación. No obstante, las limitaciones actuales de las prótesis en particular se refieren a la ausencia de capacidades sensitivas (principalmente por la carencia de receptores sensitivos o corpúsculos). Por consiguiente, el presente trabajo pretende que a través del estudio de las propiedades de los receptores sensitivos que permiten las diferentes modalidades de sensaciones somáticas se profundice en el conocimiento de los mismos, y así generar posteriores caracterizaciones que conlleven a diseños de corpúsculos artificiales; para ello se observan las causas más comunes por las que se realiza una amputación y las problemáticas más frecuentas de los usuarios de prótesis, asimismo se realiza una revisión sistemática que permita un acercamiento referente a los tipos de señal, los campos receptivos, velocidades de conducción de los corpúsculos y mecanismos en el proceso de transducción. An amputation is an event that affects the quality of life, which compromises different daily tasks, also deprives the individual of a source of information that allows him to interact with the physical world; therefore, a promising solution is rehabilitation. However, the current limitations of prostheses in particular refer to the absence of sensory abilities (mainly due to the lack of sensitive receptors or corpuscles). Therefore, the present work intends that through the study of the properties of the sensitive receptors that allow the different modalities of somatic sensations, the knowledge of them is deepened, and thus generate subsequent characterizations that lead to designs of artificial corpuscles; for this, the most common causes for which an amputation is performed and the most frequent problems of prosthesis users are observed, a systematic review is also carried out that allows an approach regarding the types of signal, the receptive fields, conduction speeds of the corpuscles and mechanisms in the transduction process. Resumen Introducción 1. Planteamiento del Problema Descripción del problema 1.1.1 Formulación del problema. 2. Justificación 3 Objetivos 3.1 Objetivo General 3.2 Objetivos Específicos 4 Estado del arte Retroalimentación sensorial 4.1.1 Codificación de datos. 4.1.2 Transmisión de datos. 4.2 Sistemas Neuromórficos 4.2.1 Modelo de Izhikevich. 4.3 Sensores 5 Marco Teórico 5.1 Amputación 5.1.1 Clasificación. 5.2 Sensación 5.2.1 El proceso de la sensación. 5.2.2 Canales iónicos y la sensación. 5.3 Sensaciones somáticas 5.3.1 Sensaciones táctiles. 5.3.2 Sensación térmica. 5.3.3 Sensación de dolor. 5.3.4 Sensación propioceptiva. 5.4 Receptores sensitivos 5.4.1 Disco de Merkel. 5.4.2 Corpúsculos de Meissner. 5.4.3 Terminaciones de Ruffini. 5.4.4 Corpúsculos de Pacini. 5.4.5 Termoreceptores. 5.4.6 Nociceptores. 5.5 La Piel 6 Metodología Tipo de investigación 6.2 Recolección de datos 6.3 Análisis de los datos 7 Resultados Esperados 8 Conclusiones Referencias Pregrado Tecnólogo en Mantenimiento de Equipos Biomédicos

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

The demand for natural products with biological application has become of great interest of the scientific community and is not different from the products produced by the bees: honey, propolis, wax and pollen. Several studies have shown the chemical composition and pharmacological properties of africanized bee propolis (Apis mellifera). However, there is a varity of more than 300 species of native bees in the Brazilian fauna, known as stingless bees (meliponineos), which few studies have been carried out, so little is known about the chemical composition and bioactivity of their products. This work aimed to investigate the chemical composition and determine the potential antimicrobial, antioxidant, antinociceptive and anti-inflammatory potential of the hydroalcoholic extract (EHP) of the propolis of Melipona quadrifasciata quadrifasciata. The chemical composition of the EHP was determined by colorimetric and chromatography (HPLC-DAD and UPLC-Q/TOF- MS/MS) analysis. Moreover, the UHPLC-MS analyses of M. quadrifasciata quadrifascita indicated as major constituent’s phenolic compounds, flavonoids and terpenes. The EHP presented antioxidant extracts presented antioxidant activity in the DPPH assay IC50= 241.8 μg/mL. The bactericidal activity was higher against gram-positive bacteria (Staphylococcus aureus, Staphylococcus aureus, meticilina resistente e Enterococcus faecalis)) than for gramnegative bacteria (Escherichia coli e Klebsiella pneumoniae). The EHP from presented MIC values lower to gram-positive bacterials than gram-negative bacterials. The EHP caused lysis of the bacterial wall and release of intracellular components in S. aureus and E. coli, suggesting that this might be one mechanism by which exerts its antibacterial effect EHP. Moreover, the hemolytic activity of S. aureus was significantly lower when incubated in the presence of sub-inhibitory concentrations of EHP. Mice treated by intragastric route with EHP (300 and 1000 mg/kg) showed a significant reduction in nociceptive behavior induced by intraplantar formalin injection, in both phases of the test. While EHP (300 mg / kg) was able to reduce nociception induced by bradykinin, prostaglandin E2, histamine, forskolin, phorbol 12-myristate 13-acetate, capsaicin, cinnamaldehyde and acidified saline. Demonstrating that EHP to modulate the activation of nociceptors by interaction direct with the TRPV1, TRPA1 and ASIC channels and by inhibiting the PKA / PKC signaling pathways. In addition, EHP treatment (300 mg/kg/i.g.) inhibited ear edema induced topically by xylene and inflammatory activity in the carrageenan-induced peritonitis test. Where the anti-inflammatory potential of EHP was established by decreased cell migration, percentage of neutrophils and protein content at the inflammatory site, as well as the decrease of the pro-inflammatory cytokines IL-17 and IL-6 and increase of the anti-inflammatory cytokine IL-10. Thus, this dataset supports, at least in part, the traditional use of the propolis ethanolic extract of Melipona quadrifasciata quadrifasciata. A procura por produtos naturais com aplicação biológica tornou-se de grande interesse da comunidade científica, e não diferentemente, ocorreu com os produtos produzidos pelas abelhas: mel, própolis, cera e pólen. Vários estudos tem sido desenvolvidos sobre a estrutura química e bioatividade da própolis de abelhas africanizadas (Apis mellifera). Contudo, há uma variedade de mais de 300 espécies de abelhas nativas na fauna brasileira, conhecidas como abelhas indígenas sem ferrão (meliponíneos), as quais poucos estudos têm sido realizados; portanto, pouco se conhece a respeito da composição química e atividade de seus produtos e subprodutos. Este trabalho se propôs a investigar a composição química e determinar o potencial antimicrobiano, antioxidante, antinociceptivo e anti-inflamatório do extrato hidroalcoólico (EHP) do própolis da Melipona quadrifasciata quadrifasciata. A análise química do EHP, através de técnicas colorimétricas e cromatográficas (HPLC-DAD and UPLC-Q/TOF- MS/MS) mostrou que os mesmos são compostos por uma mistura de polifenóis, flavonoides e terpenos. O EHP apresentou atividade antioxidante dose-dependente, sendo a IC50= 241 μg/mL. A atividade antimicrobiana foi testada contra cepas gram positivas (Staphylococcus aureus, Staphylococcus aureus, meticilina resistente e Enterococcus faecalis) e gram negativas (Escherichia coli e Klebsiella pneumoniae) através do método de microdiluição em caldo. Sendo que o EHP apresentou maior atividade antimicrobiana contra as cepas gram positivas em comparação as cepas gram negativas. O EHP causou lise bacteriana tanto em S. aureus como em E. coli, indicando que esse pode ser um dos mecanismos pelo qual o EHP exerce seu efeito antimicrobiano. Além disso, a atividade hemolítica do S. aureus foi significativamente menor quando incubado na presença de concentrações sub-inibitórias do EHP. Camundongos tratados intragastricamente (i.g.) com EHP (300 e 1000 mg/kg) apresentaram significativa redução do comportamento nociceptivo induzido pela injeção intraplantar (i.pl.) de formalina, em ambas as fases do teste. Enquanto que a dose de 300 mg/kg de EHP reduziu a nocicepção no teste da nocicepção induzida por bradicinina, prostagladina E2, histamina, forscolina, forbol-12-miristato-13-acetato, capsaicina, cinamaldeído e salina acidificada; sugerindo que o efeito antinociceptivo do EHP se dá não só por bloqueio direto dos receptores TRPV1, TRPA1 e ASIC, mas também através da inibição das vias de sinalização da PKA e PKC. O tratamento com EHP (300 mg/kg/i.g.) reduziu o edema de orelha induzido topicamente por xileno e a atividade inflamatória no teste da peritonite induzida por carragenina; onde o potencial anti-inflamatório da EHP foi estabelecido pela diminuição da migração celular, porcentagem de neutrófilos e teor de proteína no lavado peritoneal, além da diminuição das citocinas pró-inflamatórias IL-17 e IL- 6 e aumento da citocina anti-inflamatória IL-10. Assim, esse conjunto de dados suporta, pelo menos em parte, o uso tradicional do extrato hidroalcoólico de própolis da Melipona quadrifasciata quadrifasciata.

This case-control study primarily compared the trigeminal nociceptive function, the intraoral somatosensory profile and possible structural nerve changes between diabetic peripheral neuropathy (DPN, n = 12) patients and healthy participants (n = 12). The nociceptive blink reflex (nBR) was recorded applying an electrical stimulation over the entry zone of the right supraorbital (V1R), infraorbital (V2R) and mental (V3R) and left infraorbital (V2L) nerves. The outcomes were: individual electrical sensory (I0) and pain thresholds (IP); root mean square (RMS), area-under-the-curve (AUC) and onset latencies of R2 component of the nBR. Furthermore, a standardized full battery of quantitative sensory testing (QST) and intraepidermal nerve fibre density (IENFD) or nerve fibre length density (NFLD) assessment were performed, respectively, on the distal leg and oral mucosa. As expected, all patients had altered somatosensory sensitivity and lower IENFD in the lower limb. DPN patients presented higher I0, IP, RMS and AUC values (p

O sulfeto de hidrogênio (H2S) é um gás de baixa massa molecular e modula diversos processos fisiológicos e patológicos, incluindo a inflamação e a dor. Ensaios préclínicos têm demonstrado que substâncias que doam H2S inibem a liberação de mediadores inflamatórios, o recrutamento de leucócitos, a hiperalgesia e a alodínia mecânica e o edema inflamatório. As tiobenzamidas são substâncias que doam H2S e que apresentam atividades em alguns modelos experimentais. Desta forma, o presente estudo teve como objetivo avaliar as atividades da 4-metilbenzenocarbotiamida em modelos experimentais de inflamação e dores nociceptiva, inflamatória e neuropática em camundongos Swiss, bem como os possíveis mecanismos que medeiam essas atividades. A administração da 4-metilbenzenocarbotiamida (100 ou 150 mg/kg, i.p, -30 min) reduziu a resposta nociceptiva induzida por formaldeído (0,92%, 20 L, i.p.). A segunda fase da resposta nociceptiva foi inibida por ambas as doses, enquanta a primeira fase foi inibida apenas pela maior dose da 4-metil-benzenocarbotiamida. A 4-metil-benzenocarbotiamida não inibiu a resposta nociceptiva induzida por calor (modelo de placa quente, 50 ºC). A 4-metilbenzenocarbotiamida (150 mg/kg) atenuou a alodínia mecânica induzida por carragenina (600 g, 30 L, i.pl.). A atividade da 4-metil-benzenocarbotiamida nesse modelo experimental foi inibida pela administração prévia de ciproheptadina (5 ou 10 mg/kg, p.o.), mas não de naltrexona (5 ou 10 mg/kg, i.p) ou glibenclamida (20 ou 40 mg/kg, p.o). No modelo de alodínia mecânica induzida por constrição nervo isquiático, a 4-metil-benzenocarbotiamida não apresentou atividade. A atividade motora dos animais, avaliada pelo tempo de permanência na haste girante, não foi prejudicada pela administração da 4-metil-benzenocarbotiamida. A 4-metil-benzenocarbotiamida (50, 100 ou 150 mg/kg) reduziu o edema de pata, a produção de fator de necrose tumoral- e CXCL1 e a atividade da mieloperoxidase induzidos por carragenina. Foi observado, ainda, que a 4-metil-benzenocarbotiamida libera H2S in vitro. Concluindo, os resultados do presente estudo demonstram a atividade da 4-metilbenzenocarbotiamida em modelos de inflamação e dores nociceptiva e inflamatória. Os resultados indicam que a atividade da substância está relacionada com a ativação de mecanismos serotoninérgicos e a redução da produção de citocinas e do recrutamento de leucócitos. A demonstração da atividade da 4-metilbenzenocarbotiamida em modelos de dor e inflamação pode estimular a realização de estudos adicionais visando à avaliação dessa molécula como candidata a fármaco útil no tratamento de pacientes com condições dolorosas e inflamatórias Hydrogen sulfide (H2S) is a low molecular weight gas that mediates several pathophysiological processes, including inflammation and pain. Preclinical assays have demonstrated that H2S donors reduce production of inflammatory mediators, recruitment of leukocytes, edema, allodynia and hyperalgesia. Thiobenzamides are H2S donors and exhibit activities in many experimental models. Thus, the present study aimed to evaluate the activities of 4-methylbenzenecarbothioamide in experimental models of inflammation, and nociceptive, inflammatory and neuropathic pain in mice, as well as their mechanisms of action. 4-methylbenzenecarbothioamide (100 or 150 mg/kg, i.p., -30 min) reduced the nociceptive responde induced by formaldehyde. The second phase of this response was inhibited by both doses, while the first phase was inhibited only by the highest dose. 4-methylbenzenecarbothioamide did not inhibit the nociceptive response induced by heat (hot plate test, 50 ºC). Mechanical allodynia induced by carrageenan (600 g, 30 l, i.pl.) was attenuated by 4methylbenzenecarbothiamide (150 mg/kg). The effect induced by 4methylbenzenecarbothiamide in this model was inhibited by previous administration of cyproheptadine (5 or 10 mg/kg, p.o.), but not by naltrexone (5 or 10 mg/kg, i.p.) or glibenclamide (20 or 40 mg/kg, p.o.). 4-methylbenzenecarbothiamide did not inhibit mechanical allodynia induced by constriction of the sciatic nerve. The motor activity, evaluated by the time mice spent on the rota-rod, was not changed by 4methylbenzenecarbothiamide. 4-methylbenzenecarbothiamide (50, 100 or 150 mg/kg) inhibited paw edema, production of tumor necrosis factor- and CXCL1 and myeloperoxidase activity induced by carrageenan. It was also shown that 4methylbenzenecarbothiamide releases H2S in vitro. In conclusion, the results of the present study demonstrate the activity of 4-methylbenzenecarbothamide in different models of inflammation and nociceptive and inflammatory pain. The results indicate that the activity of 4-methylbenzenecarbothamide involves activation of the serotoninergic pathway and reduction of cytokines production and leukocyte recruitment. The demonstration of the activity of 4-methylbenzenecarbothamide in models of pain and inflammation may stimulate further studies aiming to evaluate this molecule as a potential candidate useful in the treatment of patients with painful and inflammatory disorders.

A dor pode ser classificada em inflamatória, neuropática e fásica, que ocorre por um dano do sistema nervoso e estimulação de nociceptores, como os Receptores de Potencial Transiente (TRP’s). Nesta família destacamos o TRPV1, TRPM8 e TRPA1, estimulados por substâncias antinociceptivas, como a capsaicina, mentol e metil salicilato, respectivamente. O trabalho objetivou investigar o papel dos TRP’s na antinocicepção, pela administração dos agonistas no acuponto Zusanli (ST36), no modelo de dor inflamatória, neuropática e fásica. Foram utilizados 180 ratos Wistars, machos, 6 semanas, entre 250 a 300g. Eles foram submetidos ao modelo de dor inflamatória induzida por Adjuvante Completo de Freund (CFA), dor fásica pelo Teste de Retirada da Cauda, Tail Flick (TF) e dor neuropática por Chronic Constriction Injury (CCI). Foi realizada uma injeção subcutânea no acuponto Zusanli de 20μL de salina e agonistas dos TRP’s, capsaicina, mentol e metil salicilato, nas doses de 0,03 μg/20μL; 0,3 μg/20μL; 3,0 μg/20μL. Os limiares mecânicos foram avaliados através do aparelho Von Frey digital, antes da administração do CFA e da cirurgia de constrição do nervo ciático, após 4 horas e 72 horas para avaliação hiperalgésica, e 0, 1, 3, 6 e 24 horas após a administração dos agonistas. E os limiares de TF foram avaliados antes e após 0, 1, 3, 6 e 24 horas. A hiperalgesia inflamatória foi revertida significativamente em todos os agonistas. A administração de capsaicina induziu redução do efeito hiperalgésico, em todos os tempos avaliados, nas 3 doses, exceto no tempo após 24 horas de tratamento, onde somente a maior dose induziu redução nos limiares mecânicos. E o tratamento com mentol e metil salicilato, no acuponto, induziu efeito antinociceptivo, em todos os tempos, nas diferentes doses. No grupo de dor fásica, a administração dos agonistas, capsaicina e mentol, apresentou aumento dos limiares de latência, em todos os tempos avaliados nas 3 doses, exceto no T0. E a administração de metil salicilato obteve efeito analgésico, em todas as doses, nos tempos T3, T6 e T24, e somente a maior dose apresentou efeito no T0 e a dose intermediária no T1. No modelo de dor neuropática, a capsaicina, nas doses maior e intermediária, apresentou efeito analgésico em quase todos os tempos. O mentol obteve este efeito em todas as doses e quase em todos os tempos, assim como o metil salicilato. De acordo com os resultados, podemos sugerir que os receptores TRPV1, TRPM8 e TRPA1 estão envolvidos no efeito anti-hiperalgésico, pela administração de agonistas no acuponto Zusanli, no controle da dor inflamatória, fásica e neuropática. Pain may be classified as inflammatory, neuropathic and phasic, which occurs by a damage in the nervous system and nociceptors stimulation, such as the Transient Receptor Potential (TRP’s). In TRP’s group, we highlight the TRPV1, TRPM8, and TRPA1, stimulated by substances anti-nociceptive, such as capsaicin, menthol, and methyl salicylate respectively. This work sought to investigate the role of the TRP’s on the antinociception, trough administration of agonists in the acupoint Zusanli (ST36), in inflammatory, neuropathic and phasic pain models. The project has composed by 180 male Wistars rats, 6 weeks old, between 250g to 300g. The inflammatory pain model induced by Complete Freund’s Adjuvant (CFA), phasic pain model by Tail Flick (TF) and neuropathic pain model by Chronic Constriction Injury (CCI). A subcutaneous injection in the acupoint Zusanli 20μL of saline and the TRP 's agonists, capsaicin , menthol and methyl salicylate was applied in doses of 0,03 μg/20μL; 0,3 μg/20μL; 3,0 μg/20μL. Mechanical thresholds assessed using a digital Von Frey device before the administration of CFA and after 0, 1, 3, 6 and 24 hours of administration of the agonists. The TF thresholds assessed before and after 0, 1, 3, 6 and 24 hours. Inflammatory hyperalgesia revealed significantly reversed in all agonists. The administration of capsaicin induced the reduction of hyperalgesic effects at all periods, in 3 doses, except for the time after 24 hours of treatment, where only the higher dose induced a reduction in a mechanical threshold. The treatment with menthol and methyl salicylate induced an antinociceptive effect in the acupoint for all the times, in different doses. In phasic pain group, the administration of agonists, capsaicin, and menthol increased the latency thresholds, at all times evaluated in three doses except the T0. In addition, the administration of methyl salicylate obtained an analgesic effect on all doses and on the times T3, T6 and T24, and only the highest dose had an effect on T0 and the intermediate dose on T1. In neuropathic model pain, capsaicin in the highest and intermediate dose showed analgesic effect in all the times. The mentol and the methyl salicylate had this effect in all the dose and the most times. According to the results obtained so far, we suggest that TRPV1, TRPM8, and TRPA1 receptors are involved in anti-hyperalgesic effect by administering agonists in the Zusanli acupoint in the control of inflammatory and phasic pain. Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

ResumenEl dolor es una experiencia cognitiva como resultado de la activación de nociceptores o descritas en esos términos. El estímulo nociceptivo desencadena la activación de redes neuronales que procesan la información en forma paralela y distribuida, con un componente discriminativo en el tálamo y corteza primaria contra lateral y otro afectivo en la corteza del cíngulo anterior y prefrontal dorsolateral. El dolor como experiencia psíquica solo puede ser objetivado en sus aspectos de activación de las áreas de la así llamada matriz de dolor, mediante la técnica de resonancia magnética funcional. Con el registro de la actividad de nociceptores en sujetos despiertos y en pacientes con dolor neuropático mediante microneurografía se puede relacionar la actividad anormal en estos receptores con la sensación evocada.SummaryPain is a cognitive experience as the consequence of activation of nociceptors, or described in such terms. A nociceptive stimulus triggers the activation of a network of parallel and distributed neural systems with a discriminative component in the thalamus and primary somatosensory cortex, and an affective component in the anterior cingulate and dorsolateral prefrontal cortex. This activity in the ‘pain matrix’ is quantitatively measured through functional MRI. Abnormal activity in primary nociceptors in awake subjects and patients with neuropathic pain is objectively related to spontaneous and evoked pain.

Gamma-aminobutyric acid (GABA)ergic neurons of the substantia nigra pars reticulata (SNpr) are connected to the deep layers of the superior colliculus (dlSC). The dlSC, in turn, connect with the SNpr through opioid projections. Nociceptin/orphanin FQ peptide (N/OFQ) is a natural ligand of a Gi protein-coupled nociceptin receptor (ORL1; NOP) that is also found in the SNpr. Our hypothesis is that tectonigral opioid pathways and intranigral orphanin-mediated mechanisms modulate GABAergic nigrotectal connections. Therefore, the aim of this work was to study the role of opioid and NOP receptors in the SNpr during the modulation of defence reactions organised by the dlSC. The SNpr was pretreated with either opioid or NOP receptor agonists and antagonists, followed by dlSC treatment with bicuculline. Blockade of GABAA receptors in the dlSC elicited fear-related defensive behaviour. Pretreatment of the SNpr with naloxone benzoylhydrazone (NalBzoH), a μ-, δ-, and κ1-opioid receptor antagonist as well as a NOP receptor antagonist, decreased the aversive effect of bicuculline treatment on the dlSC. Either μ-opioid receptor activation or blockade by SNpr microinjection of endomorphin-1 (EM-1) and CTOP promoted pro-aversive and anti-aversive actions, respectively, that modulated the defensive responses elicited by bicuculline injection into the dlSC. Pretreatment of the SNpr with the selective NOP receptor antagonist JTC801 decreased the aversive effect of bicuculline, and microinjections of the selective NOP receptor agonist NNC 63-0532 promoted the opposite effect. These results demonstrate that opioid pathways and orphanin-mediated mechanisms have a critical role in modulating the activity of nigrotectal GABAergic pathways during the organisation of defensive behaviours.

Post-ictal antinociception is characterised by an increase in the nociceptive threshold that accompanies tonic and tonic-clonic seizures (TCS). The locus coeruleus (LC) receives profuse cholinergic inputs from the pedunculopontine tegmental nucleus. Different concentrations (1μg, 3μg and 5μg/0.2μL) of the muscarinic cholinergic receptor antagonist atropine and the nicotinic cholinergic receptor antagonist mecamylamine were microinjected into the LC of Wistar rats to investigate the role of cholinergic mechanisms in the severity of TCS and the post-ictal antinociceptive response. Five minutes later, TCS were induced by systemic administration of pentylenetetrazole (PTZ) (64mg/kg). Seizures were recorded inside the open field apparatus for an average of 10min. Immediately after seizures, the nociceptive threshold was recorded for 130min using the tail-flick test. Pre-treatment of the LC with 1μg, 3μg and 5μg/0.2μL concentrations of both atropine and mecamylamine did not cause a significant effect on seizure severity. However, the same treatments decreased the post-ictal antinociceptive phenomenon. In addition, mecamylamine caused an earlier decrease in the post-ictal antinociception compared to atropine. These results suggest that muscarinic and mainly nicotinic cholinergic receptors of the LC are recruited to organise tonic-clonic seizure-induced antinociception.

The hypothalamus is a forebrain structure critically involved in the organization of defensive responses to aversive stimuli. Gamma-aminobutyric acid (GABA)ergic dysfunction in dorsomedial and posterior hypothalamic nuclei is implicated in the origin of panic-like defensive behavior, as well as in pain modulation. The present study was conducted to test the difference between these two hypothalamic nuclei regarding defensive and antinociceptive mechanisms. Thus, the GABA(A) antagonist bicuculline (40 ng/0.2 µL) or saline (0.9% NaCl) was microinjected into the dorsomedial or posterior hypothalamus in independent groups. Innate fear-induced responses characterized by defensive attention, defensive immobility and elaborate escape behavior were evoked by hypothalamic blockade of GABA(A) receptors. Fear-induced defensive behavior organized by the posterior hypothalamus was more intense than that organized by dorsomedial hypothalamic nuclei. Escape behavior elicited by GABA(A) receptor blockade in both the dorsomedial and posterior hypothalamus was followed by an increase in nociceptive threshold. Interestingly, there was no difference in the intensity or in the duration of fear-induced antinociception shown by each hypothalamic division presently investigated. The present study showed that GABAergic dysfunction in nuclei of both the dorsomedial and posterior hypothalamus elicit panic attack-like defensive responses followed by fear-induced antinociception, although the innate fear-induced behavior originates differently in the posterior hypothalamus in comparison to the activity of medial hypothalamic subdivisions.

The necessity of safe and effective treatments for chronic pain has intensified the search for new analgesic drugs. In the last few years, members of a closely-related family of ion channels, called transient receptor potential (TRP) have been identified in different cell types and their functions in physiological and pathological conditions have been characterized. The transient receptor potential ankyrin 1 (TRPA1), originally called ANKTM1 (ankyrin-like with transmembrane domains protein 1), is a molecule that has been conserved in different species during evolution; TRPA1 is a cation channel that functions as a cellular sensor, detecting mechanical, chemical and thermal stimuli, being a component of neuronal, epithelial, blood and smooth muscle tissues. In mammals, TRPA1 is largely expressed in primary sensory neurons that mediate somatosensory processes and nociceptive transmission. Recent studies have described the role of TRPA1 in inflammatory and neuropathic pain. However, its participation in cold sensation has not been agreed in different studies. In this review, we focus on data that support the relevance of the activation and blockade of TRPA1 in pain transmission, as well as the mechanisms underlying its activation and modulation by exogenous and endogenous stimuli. We also discuss recent advances in the search for new analgesic medicines targeting the TRPA1 channel.

Generalised tonic and tonic-clonic seizures are followed by significant increase in nociceptive thresholds in both laboratory animals and humans. The endogenous opioid peptides play a role in antinociceptive signalling, and the periaqueductal grey matter (PAG) is recruited to induce analgesia. Thus, the aim of this investigation was to evaluate the role of µ1 -opioid receptors in the dorsomedial (dm) and ventrolateral (vl) columns of PAG in post-ictal antinociception. Pentylenetetrazole (PTZ; 64 mg/kg), which is an ionotropic GABA-mediated Cl- influx antagonist, was intraperitoneally (IP) administered to induce tonic-clonic seizures in Wistar rats. The tail-flick test was used to measure the nociceptive threshold. Microinjections of naltrexone (5.0 µg/0.2 µL), which is a non-selective opioid receptor antagonist, in both dmPAG and vlPAG decreased the tonic-clonic seizure-induced antinociception in seizing animals from 10 to 120 min after seizures. Furthermore, microinjections of the µ1 -opioid receptor-selective antagonist naloxonazine (5.0 µg/0.2 µL) into the dmPAG decreased post-ictal antinociception immediately after convulsive reactions and from 10 to 90 min after seizures. However, vlPAG-pretreatment with naloxonazine at the same concentration decreased the post-ictal antinociception 30 min after the onset of tonic-clonic seizures and the nociceptive threshold returned to basal values 120 min after seizures. These findings indicate that µ1 -opioid receptor-signalling mechanisms in both dmPAG and vlPAG play a relevant role in the organisation of post-ictal antinociception. In addition, µ1 -opioid receptors in the dmPAG rather than in vlPAG seem to be more critically recruited during the antinociception induced by generalised tonic-clonic seizures.

Falhas na administração de fármacos pela via epidural é um problema frequentemente relatado em equinos, comparados com outras espécies. Com base em estudos com seres humanos, camundongos, cabras e cães, hipotetizou-se que neostigmina, epinefrina e suas associações à lidocaína e xilazina, pudessem intensificar e prolongar os efeitos antinociceptivos destes dois últimos fármacos, quando administrados pela via epidural em éguas. Para testar esta hipótese, o estudo foi dividido em quatro etapas, utilizando-se sempre os mesmos animais. Neostigmina, lidocaína e suas associações foram os fármacos avaliados na primeira etapa, tendo o estímulo com agulha fina o modelo de nocicepção. Como os resultados foram diferentes de estudos semelhantes, na segunda etapa testaram-se os mesmos fármacos utilizados na primeira, mas com um modelo distinto de nocicepção, a eletroestimulação. Os resultados corroboraram os encontrados na primeira etapa deste estudo. Epinefrina e neostigmina associadas ou não à lidocaína foram os fármacos avaliados na terceira etapa, utilizando-se a eletroestimulação como modelo de nocicepção. Xilazina, epinefrina, neostigmina e suas associações foram os fármacos utilizados na quarta e última etapa deste estudo, mantendo-se a eletroestimulação como modelo de nocicepção. Lidocaína foi eficiente em promover antinocicepção na região perineal por um curto período de tempo, onde a adição de neostigmina não melhorou sua ação antinociceptiva, seja no modelo de nocicepção com agulha fina ou de eletroestimulação. Epinefrina intensificou e prolongou os efeitos antinociceptivos da lidocaína, como ocorreu em estudos envolvendo seres humanos e outras espécies animais, sem desencadear efeitos adversos, conforme avaliado neste estudo. Neostigmina intensificou ainda mais os efeitos antinociceptivos da associação de epinefrina e lidocaína. Os melhores resultados encontrados neste estudo, em relação à ação antinociceptiva, foram atribuídos à xilazina, onde sua associação com neostigmina não determinou incremento antinociceptivo considerável. Número reduzido de animais, utilização de apenas uma dose de cada fármaco, além de somente dois modelos de nocicepção, foram algumas das limitações deste estudo, podendo encorajar pesquisas futuras, contribuindo com os resultados aqui encontrados. Failure to administer drugs via the epidural route is a problem more frequently reported in horses than in other species. Studies conducted on humans, mice, goats, and dogs have led to the hypothesis that neostigmine, adrenaline, and their combinations with lidocaine and xylazine may intensify and prolong the antinociceptive effects of the latter two drugs when administered to mares via the epidural route. To test this hypothesis, the current study was divided into four phases, with the same animals being used in all phases. The drugs neostigmine, lidocaine, and their combinations were evaluated in the first phase, and stimulation with a fine needle was used as the nociception model. Because the results differed from those obtained in similar studies, the same drugs were tested in the second phase but by using a different nociception model, i.e., electrostimulation. The results were in line with those obtained in the first phase of this study. The drugs evaluated in the third phase were adrenaline and neostigmine, combined or not with lidocaine, and with electrostimulation as the nociception model. Xylazine, adrenaline, neostigmine, and their combinations were used in the fourth and last phase of this study, and electrostimulation was maintained as the nociception model. Lidocaine was effective in promoting antinociception in the perineal region for a short period, during which the addition of neostigmine did not improve its antinociceptive action in both nociception models (fine needle and electrostimulation). Adrenaline intensified and prolonged the antinociceptive effects of lidocaine, as was observed in studies on humans and other animal species, without triggering any adverse effects. Neostigmine further intensified the antinociceptive effects of the combination of adrenaline and lidocaine. The best results with regard to antinociceptive action were obtained with xylazine; the combination of this drug with neostigmine did not lead to a significant increase in its antinociceptive potency. The small number of animals, use of a single dose of each drug, and use of only two models of nociception were some of the limitations of the present study. Nevertheless, these findings may provide useful information for future research.

The stimulation of the retrosplenial cortex (RSC) is antinociceptive in the rat tail-flick and formalin tests. The rat RSC is caudal to and send projections to the ipsilateral anterior cingulate cortex (ACC), which is also involved in pain processing. This study demonstrated that pre-treating the rostral (rACC), but not the caudal ACC with CoCl2 (1mM), or the rACC ablation increased the duration of the antinociceptive effect evoked by a 15-s period of electrical stimulation (AC, 60Hz, 20μA) of the RSC in the rat tail-flick. Injecting the GABA-A antagonist bicuculline (50ng/0.25μL), but not the GABA-B antagonist phaclofen (300ng/0.25μL) into the rACC also increased the duration of the stimulation-induced antinociception from the RSC. In contrast, the effects of rACC stimulation persisted after the injection of CoCl2 (1mM) into the RSC. The injection of CoCl2 into the rACC did not change the nociceptive behavior of rats during phase 1 of the formalin response but reduced licking response duration during phase 2. This effect was similar in sham or stimulated animals at the RSC. We conclude that the antinociceptive effect of stimulating the RSC in the rat tail-flick test is modulated by the rACC involving GABA-A receptors in this cortex. In contrast, the antinociceptive effect of stimulating the RSC in the formalin test does not involve the rACC.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas Submitted by azevedo@ufp.pt (azevedo@ufp.pt) on 2013-10-24T14:22:24Z No. of bitstreams: 1 CláudiaVarandasPPG.docx: 741668 bytes, checksum: 7d8436303a2cb3fc60c70c97e0db56ae (MD5) Approved for entry into archive by csousa@ufp.pt (csousa@ufp.pt) on 2013-10-28T12:14:33Z (GMT) No. of bitstreams: 1 CláudiaVarandasPPG.docx: 741668 bytes, checksum: 7d8436303a2cb3fc60c70c97e0db56ae (MD5) Made available in DSpace on 2013-10-28T12:14:34Z (GMT). No. of bitstreams: 1 CláudiaVarandasPPG.docx: 741668 bytes, checksum: 7d8436303a2cb3fc60c70c97e0db56ae (MD5) Previous issue date: 2013

Several reports have shown that cutaneous leishmaniasis lesions are painless, suggesting that Leishmania infection interferes with pain perception. Comparisons of inflammation-induced hyperalgesia between BALB/c and C57BL/6 mice have been little explored in the literature, and comparative data regarding nociception in leishmaniasis are non-existent. In susceptible BALB/c mice and resistant C57BL/6 mice that were intradermally inoculated with a low dose of Leishmania major in the ear, we investigated the variation in nociception over a 12-wk period post-infection and this variation’s association with the structure of nerve fibres and the presence of endogenous cytokines that are classically considered hyper- or hypo-nociceptive. Infected BALB/c mice presented susceptibility and severe lesions. Infected C57BL/6 mice exhibited resistance and healing lesions. The immune response involved pro- and anti-inflammatory cytokine secretion, respectively. The infection-induced hypoalgesia in BALB/c mice after wks 9 was accompanied by decreased levels of IL-6 and IL-10 in ear tissue with intact nerves. C57BL/6 mice showed short-lived hyperalgesia in wks 2, which was related to increased local levels of IL-6, KC/CXCL-1, TNF-α and IL-10 and a decrease in nerve density. The increase in pro-inflammatory cytokine IL-6, KC/CXCL-1 and TNF-α levels during hyperalgesia suggested a role for these mediators in afferent nerve sensitisation, which was secondary to the inflammatory damage of nerve fibres stained by PGP 9.5.In contrast, the mechanisms of hypoalgesia may include the downregulation of cytokines, the preservation of the structure of nerve endings, and as yet uninvestigated unidentified differences in neurotransmitter release or a direct role of the parasites in the context of the progressive and permissive inflammatory response of BALB/c mice.

Orientadores: Carlos Amilcar Parada, Helder José Ceragioli Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Nanopartículas são estruturas com dimensões nanométricas que vem demonstrando um potencial para várias aplicações, inclusive na área biológica. De acordo com o seu processo de síntese e crescimento estas partículas podem possuir propriedades magnéticas com maior ou menor magnitude. O objetivo deste trabalho foi avaliar se a administração no espaço sub-aracnóide da medula espinal, de nanopartículas magnéticas altera o limiar de resposta a estímulos mecânicos aplicados em patas de ratos. As nanopartículas de carbono (nanotubos ou nanofibras) foram caracterizadas através do espectro de Raman e o seu comportamento magnético caracterizado pelo ferromagnetismo foi avaliado através da curva de histerese. As nanopartículas foram administradas via intratecal em ratos Wistar machos levemente anestesiados com Isoflorano. A resposta comportamental ao estímulo mecânico foi avaliada por um analgesímetro eletrônico (Von Frey eletrônico) que mede o limiar mecânico de retirada da pata (nocicepção mecânica). As nanopartículas foram classificadas de acordo com o comportamento magnético em: alto magnetismo e baixo magnetismo. A administração de nanopartículas (5, 20 ou 80 ?g) com alto magnetismo, mas não com baixo magnetismo, diminuiu o limiar mecânico de retirada da pata, quando comparada com a administração do seu solvente propilenoglicol (20?L). A diminuição do limiar mecânico foi observada nos tempos de 15, 30 e 60 minutos após a administração de 20?g de nanopartículas com alto magnetismo, sendo que após este período as respostas retornaram aos seus limiares basais. Por outro lado, a administração intraplantar das nanopartículas não alterou o limiar mecânico quando comparados com as respostas basais. Portanto, a administração na medula espinal, mas não no tecido periférico, de nanopartículas com alto magnetismo diminuiu o limiar mecânico. Em conclusão, os dados deste trabalho sugerem que a transmissão neuronal na medula espinal envolve uma atividade eletromagnética Abstract: Nanoparticles are structures with nanometer dimensions that are emerging to various applications, including that one involved in biological area. According to their synthesis and growth techniques these particles can have magnetic properties with greater or lesser magnitude. The objective of this study was to evaluate whether the magnetic nanoparticles administration in the subarachnoid space of the spinal cord induces changes in the mechanical nociceptive threshold in rats hind-paw. The carbon nanoparticles (nanotubes or nanofibers) were characterized by Raman spectrum and their magnetic characterized by the ferromagnetism behavior was evaluated by the hysteresis curve. The nanoparticles were intrathecally administered in Wistar rats lightly anesthetized with Isoflorano. The behavioral response to mechanical stimulation was assessed by an electronic analgesimeter (electronic Von Frey) that measures the withdrawal mechanical threshold (mechanical nociception). The nanoparticles were classified according to their magnetic behavior in: high magnetism and low magnetism. The administration (5, 20 or 80 ?g) of high magnetism nanoparticles, but not of low magnetism nanoparticles, decreased the mechanical withdrawal threshold of the hind-paw, when compared with the administration of propylene glycol (20 ?L). Decreasing in mechanical withdrawal threshold was observed at 15, 30 and 60 minutes after administration of 20?g of high magnetism nanoparticles. After this period, the mechanical withdrawal thresholds returned to their normal baseline. In contrast, intraplantar administration of the nanoparticles did not alter the mechanical withdrawal threshold. Therefore, administration of high magnetic nanoparticles in the spinal cord, but not in peripheral tissue, decreases the mechanical withdrawal threshold. In conclusion, our data suggest that neuronal transmission in the spinal cord involves an electromagnetic activity Mestrado Fisiologia Mestre em Biologia Funcional e Molecular

Fil: Srinivasan, Venkataramanujan. Sri Sathya Sai Medical Educational and Research Foundation; India Fil: Pandi Perumal, Seithikurippu R. Somnogen Inc.; Estados Unidos Fil: Moscovitch, Adam. Investigador Independiente; Canadá Fil: Spence, David Warren. Investigador Independiente; Canadá Fil: Trakht, Ilya. Universidad Columbia. Colegio de Médicos y Cirujanos. Departamento de Medicina. División de Farmacología Clínica y Terapéutica Experimental; Estados Unidos Fil: Brown, Gregory M. Universidad de Toronto. Departamento de Psiquiatría; Canadá Fil: Cardinali, Daniel Pedro. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Departamento de Docencia e Investigación; Argentina abstract: Melatonin is a remarkable molecule with diverse physiological functions. Some of its effects are mediated by receptors while other, like cytoprotection, seem to depend on direct and indirect scavenging of free radicals not involving receptors. Among melatonin´s many effects, its antinociceptive actions have attracted attention. When given orally, intraperitoneally, locally, intrathecally or through intracerebroventricular routes, melatonin exerts antinociceptive and antiallodynic actions in a variety of animal models. These effects have been demonstrated in animal models of acute pain like the tail-flick test, formalin test or endotoxin-induced hyperalgesia as well as in models of neuropathic pain like nerve ligation. Glutamate, gamma-aminobutyric acid, and particularly, opioid neurotransmission have been demonstrated to be involved in melatonin´s analgesia. Results using melatonin receptor antagonists support the participation of melatonin receptors in melatonin´s analgesia. However, discrepancies between the affinity of the receptors and the very high doses of melatonin needed to cause effects in vivo raise doubts about the uniqueness of that physiopathological interpretation. Indeed, melatonin could play a role in pain through several alternative mechanisms including free radicals scavenging or nitric oxide synthase inhibition. The use of melatonin analogs like the MT1/MT2 agonist ramelteon, which lacks free radical scavenging activity, could be useful to unravel the mechanism of action of melatonin in analgesia. Melatonin has a promising role as an analgesic drug that could be used for alleviating pain associated with cancer, headache or surgical procedures.

Lectins are non-immune (glyco)proteins that can recognize and reversibly bind to carbohydrates or other substances derived from sugars. Lonchocarpus sericeus seeds (LSL) shows a molecular mass of 23555 ± 15 Da and a a-metyl glucopyranoside and N-acetyl-glucosamine binding specificity. The aim of this work was to evaluate the antinoceptive and antiinflammatory activity of lectin from Lonchocarpus sericeus seeds (LSL). To do so, we used swiss mice (25-35g). In the antiinflammatory activity studying, LSL (10 or 100 mg/kg; i.v.; 15 minutes) inhibited the migration (neutrophil rolling and adhesion) to the peritoneal cavities of animals stimulated with carrageenan (Cg), and the effect seems to be related to the cytokines (TNF-a and IL-1b) and chemokines (MIP-1a [CCL3], KC [CXCL1]) level reduction induced by this lectin. The LSL inhibitory effect on the neutrophil migration seems to be also related to the nitric oxide (NO) systemic levels increasing. LSL was effective on the NO increasing on animals’ serum. The pretreatment of mice using aminoguanidin (nitric oxide reduced synthase inhibitor) reversed the antiinflammatory effect of lectin on the neutrophil migration. Still referring to the anti-inflammatory activity, LSL was effective on inhibiting the neutrophil migration to the peritoneal cavities of animals immunized and stimulated with ovalbumin (OVA), but the lectin was unable to inhibit an in vitro neutrophil migration induced by MIP-2, showing an indirect action of LSL (cytokines and chemokines) on the migration inhibition. In the antinoceptive study, LSL (3 or 10 mg/kg; i.v.; 30 minutes) reduced abdominal contortion induced by acetic acid and only decreased the second phase of formalin test, showing an activity on the inflammatory pain. In the hot plate test LSL didn’t show any effects. To evaluate the anti-inflammatory pain, we did a study based on the anthihypernociceptive activity. Thus, LSL (10 or 100 mg/kg; i.v.; 15 minutes) inhibited the mechanical hypernociception induced by carrageenan and ovalbumin (immunized animals) intraplantarly administration, but not induced by prostaglandin E2. (PGE2). This effect was correlated to the block of neutrophil influx, suggested by the reduction of myeloperoxidase (MPO) levels on animal’s paws pretreated with LSL (3 or 10 mg/kg; i.v.; 15 minutes) and stimulated with carregeenan. This antihyperalgesic effect seems to be highly dependent of the cytokines (TNF-a and IL-1b) and chemokines (MIP-1a [CCL3], KC [CXCL1]) level decreasing, since LSL (10 or 100 mg/kg; i.v.; 15 minutes) reduced these mediators’ levels on animal’s paws pretreated with Cg (intraplantar). Corresponding to the possible central activity, LSL (10 mg/kg; i.v.) did not alter the motor activity and not provoked depression on animals. LSL acute toxicity was evaluated by treating the rats with LSL (10 mg/kg; i.v.) during seven days analyzing several parameters of the animals: kidney functions (wet weight, urea dosage), liver functions (wet weight, kinetic evaluation of aspartate aminotransferase and alanine aminotransferase), heart (wet weight) stomach (wet weight and visual evaluation of possible lesions), variation in body weight of treated animals and leukogram. The obtained results didn’t show any alteration on the evaluated parameters suggesting that LSL doesn’t show any toxicity on animals. In conclusion, the antinoceptive and antihyperalgesic activity of LSL are associated to the inhibition of the neutrophil migration; it is probably a reflection of an inhibition on cytokines and chemokines free flowing and for the increasing of NO free flow. Additionally, this lectin doesn’t show acute toxicity and central effects. Lectinas são (glico)proteínas de origem não imune e que podem reconhecer e se ligar reversivelmente a carboidratos ou a outras substâncias derivadas de açúcares. A lectina de sementes de Lonchocarpus sericeus (LSL) apresenta massa molecular aparente de 23555 ± 15 Da e especificidade de ligação a N-acetil-glicosamina e α -metil-glicopiranosídeo. O objetivo deste trabalho foi avaliar a atividade antiinflamatória e antinociceptiva da Lectina de sementes de Lonchocarpus sericeus (LSL). Para tal, utilizamos camundongos Swiss albinos (25-35g). No estudo da atividade antiinflamatória, LSL (3 ou 10 mg/kg; e.v.; 15 minutos) inibiu a migração (rolamento e adesão de neutrófilos) para a cavidade abdominal de animais estimulados com carragenina (Cg), e este efeito parece estar relacionado com a redução dos níveis de citocinas (TNF-α and IL-1β) e quimiocinas (MIP-1α [CCL3], KC [CXCL1]) por esta lectina. O efeito inibitório da LSL sobre a migração de neutrófilos, parece também envolver o aumento nos níveis sistêmicos de óxido nítrico (NO), pois LSL (10 mg/kg; e.v.) foi capaz de aumentar os níveis de NO no soro de animais, e o pré-tratamento de camundongos com aminoguanidina (inibidor da óxido nítrico sintase induzida) foi capaz de reverter o efeito aniinflamatório desta lectina sobre a migração de neutrófilos. Ainda em relação à atividade antiinflamatória, LSL foi capaz de inibir a migração de neutrófilos para a cavidade peritoneal de animais imunizados e estimulados com ovoalbumina (OVA), porém, esta mesma lectina foi ineficaz em inibir a migração de neutrófilos in vitro estimulada por MIP-2, demonstrando então um papel indireto da LSL (redução de citocinas e quimiocinas) na inibição da migração. No estudo da atividade antinociceptiva, LSL (10 ou 100 mg/kg; e.v.; 15 minutos) reduziu as contorções abdominais induzidas por ácido acético e diminuiu somente a segunda fase do teste da formalina, demostrando uma atividade sobre a dor inflamatória. No teste da placa quente, LSL não apresentou efeito. Para avaliar a dor inflamatória realizaram-se estudos de atividade antihipernociceptiva. Assim, LSL (3 ou 10 mg/kg; e.v.; 15 minutos) inibiu a hipernocicepção mecânica induzida por administração intraplantar de carragenina e ovalbumina (animais imunizados) mas não a induzida por prostaglandina E2 (PGE2). Este efeito foi correlacionado ao bloqueio do influxo de neutrófilos, sugerido pela redução dos níveis de mieloperoxidase (MPO) nas patas de animais pré-tratados com LSL (3 ou 10 mg/kg; e.v.; 15 minutos) e estimulados com carragenina. Este efeito antihiperalgésico parece também depender da redução dos níveis de citocinas (TNF-α and IL-1β) e quimiocinas (MIP-1α [CCL3], KC [CXCL1]), uma vez que LSL (3 ou 10 mg/kg; e.v.; 15 minutos) reduziu os níveis destes mediadores no tecido da pata de animais estimulados com Cg (intraplantar). Em relação a uma possível atividade central, LSL (10 mg/kg; e.v.) não alterou a atividade motora e nem provocou depressão nos animais. A toxicidade aguda foi avaliada pelo tratamento de camundongos com LSL (10 mg/kg; e.v.), durante sete dias consecutivos, através de vários parâmetros: funções do rim (peso úmido, dosagem de uréia) e do fígado (peso úmido, avaliação da cinética da aspartato amino transaminase e alanina amino transaminase), coração (peso úmido), estômago (peso úmido e avaliação visual de possíveis lesões), variação de massa corporal dos animais tratados e leucograma. Os resultados obtidos não mostraram qualquer alteração dos parâmetros avaliados, demonstrando que a LSL não apresenta nenhuma toxicidade nos animais. Em conclusão, a atividade antiinflamatória e antinociceptiva da LSL está associada com a inibição da migração de neutrófilos, que provavelmete é reflexo da inibição da liberação de citocinas e quimiocinas e do aumento na liberação de NO. Adicionalmente, esta lectina não apresenta efeitos centrais nem toxicidade aguda.

A Dor Neuropática é definida como dor causada por lesão ou disfunção do sistema nervoso, como resultado da ativação anormal da via nociceptiva (fibras de pequeno calibre e trato espinotalâmico). As principais causas desta síndrome são: diabetes melito, neuralgia pós-herpética, neuralgia trigeminal, dor regional complexa, acidente vascular encefálico, esclerose múltipla, lesão medular, entre outros. Nos últimos anos, a Dor Neuropática vem recebendo especial atenção por dois motivos principais: 1) refratariedade terapêutica de várias síndromes dolorosas com componentes neuropáticos predominantes e 2) desenvolvimento de ferramentas diagnósticas para o reconhecimento deste tipo de dor. O presente artigo de revisão fornece informações relevantes para o entendimento e reconhecimento da Dor Neuropática, bem como quanto à abordagem terapêutica baseada em evidências. Neuropathic Pain is defined as pain caused by lesion or dysfunction of the nervous system, as a result of abnormal activation of the nociceptive pathway (small fibers and spinothalamic tracts). The most common causes of this syndrome are the following: diabetes, post-herpetic neuralgia, trigeminal neuralgia, complex regional pain syndrome, stroke, multiple sclerosis, spinal cord injury, among others. In the last few years, the Neuropathic Pain, has been receiving special attention for two main reasons: 1) therapeutical refractoriness of a variety of pain syndromes with predominant neuropathic characteristics and 2) the development of diagnostic tools for Neuropathic Pain complaints. The present paper provides relevant information on the understanding and recognition of Neuropathic Pain, as well as therapeutic evidence-based-medicine approaches.

El dolor es un signo de enfermedad y un motivo frecuente de consulta; se clasifica en agudo o crónico, nociceptivo o neuropático, y según la velocidad de conducción en rápido o lento. Los estímulos causantes del dolor son detectados por receptores nociceptores; los cuales son identificados como fibras C y fibras Aδ. El proceso neural de la transmisiσn del dolor comprende: La transducciσn; es el proceso por el cual el estνmulo nociceptivo es convertido en seρal elιctrica en los nociceptores. La transmisiσn; es el proceso por el cual los estímulos nociceptivos son referidos al asta dorsal de la medula espinal, donde se liberan los neurotransmisores del dolor: Glutamato, sustancia P, péptido relacionado al gen de la calcitonina. Seguidamente el estímulo cruza al lado contralateral de la medula espinal y viaja en el haz espinotálamico hasta el tálamo y luego a la corteza cerebral. La modulación; es el proceso por el cual la señal nociceptiva en el asta dorsal de la medula puede ser inhibida y modificada para los centros superiores del dolor. Los opiodes endógenos y exógenos dan lugar a un bloqueo indirecto de los canales de calcio y apertura de los canales de potasio, con hiperpolarización celular e inhibición de la liberación de mediadores del dolor. La activación del sistema neural descendente da lugar a la liberación de b endorfinas, encefalinas, dinorfinas; que alivian el dolor. Pain is a sign of illness and a common complain that bring patients to a health care facility. It is classified in acute or chronic pain, nociceptive or neuropathic, and according to conduction velocity, fast or slowly. C and Aδ- fibers respond to stimuli and produce the experience of pain, this defines them as nociceptors. Central pathways of pain include: nociceptors respond to stimuli and produce the experience of pain when they are electrically stimulated (Transduction); axons of nociceptors enter the spinal cord via the dorsal root, where Glutamate are Substance P are released, spinal neurons send their axons to the contralateral thalamus (Transmission); the perceived intensity of pain can be modulated by brain circuits (Modulation). Pain inhibition is achieved by calcium channel blockage and open potassium channel by endogenous and exogenous opioids, due hyperpolarization of the cell and inhibition of pain mediators. b endorphins, enkephalins and dinorphins are released due to activation of neural descending pathway causing pain relief.

Premio Extraordinario de Doctorado 2009