Orientador: João Ernesto de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T01:37:30Z (GMT). No. of bitstreams: 1 Figueiredo_MarianaCecchetto_M.pdf: 1623001 bytes, checksum: 8e3ade8e07506ea16870f8811c85daa3 (MD5) Previous issue date: 2013 / Resumo: Este trabalho teve como objetivo realizar uma triagem inicial da ayahuasca em cultura de células e em modelos de nocicepção e ansiedade. A ayahuasca é uma decocção de duas plantas (Banisteriopsis caapi e Psychotria viridis) com ação alucinógena, utilizada por diversas religiões no Brasil. As principais substâncias relacionadas com as atividades no sistema nervoso central são a DMT(dimetiltriptamina), agonista serotoninérgico dos receptores 5HT1A, 2A/2C e as ?-carbolinas, inibidoras da monoaminoxidase-A (MAO), encontradas na P. viridis e B. caapi, respectivamente. Nos últimos anos, a ayahuasca se espalhou pela Europa e Estados Unidos, chamando a atenção dos pesquisadores quanto aos seus efeitos. Desta forma, o presente trabalho revelou que a ayahuasca possui atividade sobre a nocicepção em modelos de contorção abdominal e placa quente, atividade ansiolítica em ratos submetidos aos testes de labirinto em cruz elevado (LCE) e efeito citostático leve em cultura de células tumorais humanas de mama (MCF-7), rim (786-0), pulmão (NCI-H460), próstata (PC-3), ovário (OVCAR-3), cólon (HT-29), mama resitente a múltiplas drogas (NCI-ADR) / Abstract: This study aimed to perform an initial screening of ayahuasca in cultured cells and in models of nociception and anxiety. Ayahuasca is a decoction of two plants (Banisteriopsis caapi and Psyvhotria viridis) with hallucinogenic action, used by various religions in Brazil. The main activities related substances in the central nervous system are DMT ( dimethyltryptamine ) , serotonin 5HT1A receptor agonist, 2A/2C and ? - carbolines , inhibiting monoamine oxidase -A ( MAO ) , found in P. viridis and B. caapi , respectively. In recent years, ayahuasca has spread to Europe and the United States, calling the attention of researchers for their effects. Thus , the present study showed that ayahuasca has activity on nociception in models writhing and hot plate , anxiolytic activity in rats submitted to the elevated maze tests (EPM ) and light cytostatic effect on cultured human tumor cells breast ( MCF -7) , kidney ( 786-0 ) , lung ( NCI- H460 ) , prostate (PC -3), ovarian ( OVCAR -3) , colon ( HT -29) , breast , multidrug resistente ( NCI- ADR ) / Mestrado / Ensino em Saúde / Mestra em Clínica Médica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pain is the most common complaint in the medical field and the identification of compounds that can effectively treat painful states without induction of side-effects remains a major challenge in biomedical research. The aim of the present study was to investigate the antinociceptive effect of a novel compound, 3-(4-fluorophenyl)-5-trifluoromethyl-1H-1-tosylpyrazole (compound A) in several models of pain in mice and compare with those produced by the known trifluoromethyl-containing pyrazole compound celecoxib. Compound A or celecoxib were administrated by oral (78 780 μmol/kg), intrathecal (9 22.5 nmol/site) or intracerebroventricular (9 22.5 nmol/site) routes. Oral administration of either compound A or celecoxib abolished the mechanical allodynia, but not the oedema caused by intraplantar injection of carrageenan. Similarly, compound A reduced the overt nociception, but not the oedema, produced by bradykinin or capsaicin. However, compound A (500 μmol/kg, orally) did not alter nociception nor oedema caused by intraplantar injection of prostaglandin E2 or glutamate, whereas celecoxib reduced only the nociception induced by the former. Moreover, oral and intrathecal administration of compound A or celecoxib also reduced the nociception induced by acetic acid. However, only celecoxib reduced the acetic acid-induced nociception when it was injected by the intracerebroventricular route. Finally, neither compound A nor celecoxib was able to produce antinociceptive effect in the tail-flick test or to alter the motor performance and the body temperature. Besides, compound A or celecoxib did not induce gastric lesion. Thus, compound A seems to be an interesting prototype for the development of novel analgesic drugs. / A dor é uma das maiores queixas na área médica e a identificação de compostos que possam tratar efetivamente os estados dolorosos, sem induzir efeitos colaterais, permanece um grande desafio na pesquisa biomédica. O objetivo do presente estudo foi investigar o efeito antinociceptivo de um novo composto 3-(4-fluorfenil)-5-trifluormetil-1H-1-tosilpirazol (composto A) em diferentes modelos de dor em camundongos e comparar os seus efeitos com aqueles produzidos por um pirazol contendo trifluormetil em sua estrutura, o celecoxibe. O composto A ou o celecoxibe foram administrados por via oral (78 780 μmol/kg), intratecal (9 22,5 nmol/sitio) ou intracerebroventricular (9 22,5 nmol/sitio). A administração oral do composto A ou do celecoxibe aboliram a alodínia mecânica, mas não o edema, causado pela injeção intraplantar de carragenina. Do mesmo modo, a administração do composto A reduziu a nocicepção, mas não o edema, produzido pela bradicinina ou capsaicina. Entretanto, o composto A administrado por via oral (500 μmol/kg) não alterou a nocicepção nem o edema causados pela injeção intraplantar de prostaglandina E2 (PGE2) ou glutamato. Já o celecoxibe, reduziu somente a nocicepção induzida pela PGE2. Além disso, a administração oral ou intratecal do composto A ou celecoxibe também reduziu a nocicepção induzida por ácido acético. Quando administrados por via intracerebroventricular, somente o celecoxibe foi capaz de reduzir a nocicepção induzida por ácido acético. Finalmente, o composto A ou o celecoxibe não alteraram a nocicepção térmica aguda, a performance motora ou a temperatura corporal dos animais, nem produziram lesões gástricas quando administrados por via oral. Consequentemente, o composto A parece ser um interessante protótipo para o desenvolvimento de novas drogas analgésicas.

(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...

Spinal cord stimulation (SCS) inhibits substance P (SP) release and decreases the expression of the transient receptor potential vanilloid 1 (TRPV1) in the spinal cord at thoracic 4 (T4) during cardiac ischemia in rat models (. Ding et al., 2007). We hypothesized that activation of TRPV1 in the T4 spinal cord segment by intermittent occlusion of the left anterior descending coronary artery (CoAO) mediates spinal cord SP release. Experiments were conducted in urethane-anesthetized Sprague-Dawley male rats using SP antibody-coated microprobes to measure SP release at the central terminal endings of cardiac ischemic-sensitive afferent neurons (CISAN) in the spinal T4 dorsal horns. Vehicle, capsaicin (CAP; TRPV1 agonist) and capsazepine (CZP; TRPV1 antagonist) were injected into the left T4 prior to stimulation of CISAN by intermittent CoAO (with or without upper cervical SCS). CAP induced endogenous SP release from laminae I and II in the T4 spinal cord above baseline. Conversely, CZP injections significantly inhibited SP release from laminae I-VII in the T4 spinal cord segment below baseline. CZP also attenuated CoAO-induced SP release, while T4 injections of CZP with SCS completely restored SP release to basal levels during CoAO activation. CAP increased the number of c-Fos (a marker for CISAN activation) positive T4 dorsal horn neurons compared to sham-operated animals, while CZP (alone or during CoAO and SCS. +. CoAO) significantly reduced the number of c-Fos positive neurons. These results suggest that spinal release of the putative nociceptive transmitter SP occurs, at least in part, via a TRPV1 mechanism.

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6-38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-D-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05-0.5μg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6-38) (0.5-2μg) or PACAP-antiserum (1:500-2000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors.

Indiana University-Purdue University Indianapolis (IUPUI) / Damage Associated Molecular Patterns (DAMPs) act largely as endogenous ligands to initiate and maintain the signaling of both inflammatory processes and the acquired immune response. Prolonged action of these endogenous signals are thought to play a significant role sterile inflammation which may be integral to the development of chronic inflammation pathology. HMGB1 (High Mobility Group Box 1) is a highly conserved non-acetylated protein which is among the most important chromatin proteins and serves to organize DNA and regulate transcription. Following stress or injury to the cell, hyperacetylation of lysine residues causes translocation of HMGB1 and eventual release into the extracellular environment where it can take the form of a DAMP and interact with cell types bearing either the Receptor for Advanced Glycation End-products (RAGE) or Toll-Like Receptor 4 (TLR4). Activation of these surface receptors contribute directly to both acute and chronic inflammation. This project investigated the role of HMGB1 through its receptors Receptor for Advanced Glycation End-products (RAGE) and Toll-Like Receptor 4 (TLR4) as it pertained to the development of chronic inflammation and pathology in small diameter, nociceptive sensory neurons. It was demonstrated that the neuronal signaling associated with exposure to HMGB1 is dependent upon the ligands conformational states, as the state dictates its affinity and types of neuronal response. Neuronal activation by bacterial endotoxin or the disulfide state of HMGB1 is dependent on TLR4 and the associated signaling adapter protein, Myeloid differentiation primary response gene 88 (MYD88). Interruption of the receptor-mediated signaling cascade associated with MyD88 was shown to be sufficient to mitigate ligand-dependent neuronal activation and demonstrated significant behavioral findings. Further downstream signaling of HMGB1 in the neuron has yet to be identified, however important steps have been taken to elucidate the role of chronic neuroinflammation with hopes of eventual translational adaptation for clinical therapeutic modalities.

Nociception is the sensory nervous system that detects harmful stimuli including excessive heat, cold, toxic chemicals, and noxious mechanical stimulations. Transient receptor potential (TRP) channels are a group of evolutionarily conserved ion channels consisting of 4 subunits, each with 6 transmembrane spans, and detect a variety of external and internal nociceptive stimuli. Due to their critical roles in nociception, it is essential to understand the mechanisms that regulate TRP channels and subsequent nociception. Here, I investigated two distinct types of regulation of Drosophila transient receptor potential cation channel A1 (TrpA1): regulation via the expression of different TrpA1 isoforms, and via its binding with associated proteins. I found that one of the TrpA1 isoforms, TrpA1(E), inhibits the thermal responses of other TrpA1 isoforms in vitro. I also identified potential TrpA1 binding partners through Co- immunoprecipitation (Co-IP) and mass spectrometry analysis. These binding partners need further validation and characterization through biochemical, cellular, and behavioral assays to illustrate their roles in nociception, and may serve as potential drug targets for chronic pain.

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