Emerging evidence suggests that opioid drugs can exacerbate neuroAIDS. Microglia are the principal neuroimmune effectors thought to be responsible for neuron damage in HIV-infected individuals, and evidence suggests that drugs acting via opioid receptors in microglia aggravate the neuropathophysiological effects of HIV. The P2X family of ATP activated ligand-gated ion channels regulates key aspects of microglial function. In addition, opioid-dependent microglial activation has been reported to be mediated through P2X4 signaling, prompting us to investigate P2X receptors contribution to the neurotoxic effects of HIV and morphine. In vitro experiments showed treatment with TNP-ATP prevented the neurotoxic effects of morphine and/or HIV Tat, or ATP alone in a concentration dependent manner. This evidence suggests P2X receptors mediate the neurotoxic effects of these insults in striatal neurons. P2X1, P2X3, and P2X7 selective receptor antagonists did not prevent Tat- and/or morphine-induced neurotoxicity, implying cellular pathways activated may not involve these subtypes. Cells from P2X4KO mice show that activation of the P2X4 receptor on glia are necessary to cause Tat and/or morphine toxicity. However, data implied that baseline neuronal function may be altered due to lack of P2X4 receptor expression, and also gave evidence for altered Tat and morphine cellular signaling when the two are given in combination versus alone. Surgeries were performed on P2X4 KO and WT mice, which received intrastriatal Tat injections and morphine and/or naltrexone pellets. WT mice showed significant increases in inflammatory markers when treated with Tat and/or morphine. Increases in inflammatory markers were not seen in P2X4 KO mice, implying P2X4 receptors play a role in neuroinflammation resulting from Tat and/or morphine. Finally, human tissue samples from the National NeuroAIDS Tissue Consortium were analyzed. Changes in P2X5 and P2X7 mRNA were found in microarray data, but only changes in P2X7 mRNA levels were confirmed by RT-PCR. No changes in P2X4 mRNA levels were detected. Our experiments indicate the P2X receptor family contributes to Tat- and morphine- related neuronal injury, and reveal that members of the P2X receptor family, especially P2X4, may be novel therapeutic targets for restricting the synaptodendritic injury and neurodegeneration that accompany neuroAIDS and opiate abuse.

Background – An increase in opioid prescribing has led to an increase in opioid overdoses.1,2 No study has estimated the incremental costs subsequent to an opioid overdose event in prescription opioid users, or the prevalence and costs of overdose events in family members of prescription opioid users and in overdose victims with no identifiable source of prescription opioid. The latter group will be referred to as “others”. Objectives – The first objective of this study was to estimate the prevalence of opioid overdoses in aforementioned groups. The second objective was to estimate the incremental costs and resource utilization associated with opioid overdoses in these groups. Methods – This study is a retrospective analysis using claims data from SelectHealth, a not-for-profit health insurance organization in Utah and southern Idaho. We estimated the prevalence of opioid overdoses in the sample population, as well as in each group, by year. For the cost estimation we collapsed family members and others into one category – “non-medical users”. To estimate costs we used an incremental cost approach whereby we used propensity scores to match cases (patients who suffered from an opioid overdose) to appropriate controls (patients who did not suffer from an opioid overdose) and estimated the direct medical costs incurred in each group in the year following an overdose. Generalized Linear Models were used to estimate incremental costs and resource utilization. Sensitivity analyses were conducted to measure the robustness of the estimates. Results – The prevalence of opioid overdoses increased by 84.8% in prescription opioid users (from 55.6 per 100,000 in 2011 to 102.8 per 100,000 in 2014), increased by 37.9% in family members of prescription opioid users (from 5.9 per 100,000 in 2011 to 8.2 per 100,000 in 2014) and increased by 179.9% in others (from 8.2 per 100,000 in 2011 to 23.1 per 100,000 in 2014). The prevalence of opioid overdoses in acute users increased by 14.7% (from 43.8 per 100,000 in 2011 to 50.3 per 100,000 in 2014) as compared to 165.9% in chronic users (from 187.0 per 100,000 in 2011 to 497.3 per 100,000 in 2014). The incremental direct medical costs per patient per year were estimated to be $65,277 (p-value<0.05) in prescription opioid users who suffered from an overdose and $41,102 (p-value<0.05) in non-medical users who suffered from an overdose. Overdose-specific costs were estimated to be $12,111 for prescription opioid users and $11,070 in non-users. Conclusions – Our study found that the prevalence of opioid overdoses increased steadily from 2011 to 2014 in the sample population. The prevalence of overdoses was much higher in chronic opioid users as compared to acute users. Differences between overdose-specific costs and total incremental costs may suggest that overdoses are associated with substantial costs in addition to costs for the initial treatment of the overdose. While the cost to payers due to overdoses in prescription opioid users is substantial, payers also incur costs from diversion of opioids.

Existe um grande interesse científico voltado para a busca das bases neuropsicofarmacológicas dos comportamentos que têm sido associados ao medo e ao pânico. Muitos estudos sugerem o teto mesencefálico (TM) como responsável pelo controle de respostas defensivas elaboradas durante situações de perigo iminente. A substância cinzenta periaquedutal (SCP), as camadas profundas do colículo superior (cpCS) e o colículo inferior (CI) têm sido considerados importantes estruturas na elaboração do medo inato e do comportamento de defesa, assim como na organização da antinocicepção induzida pelo medo inato. Contudo, muitos estudos têm implicado a via neoestriado-nigro-tectal no controle de respostas defensivas elaboradas no mesencéfalo dorsal, permeadas pela interação entre vias opioides e GABAérgicas. O presente trabalho tem por objetivo o estudo neurofarmacológico da anatomia conectiva funcional entre a substância negra (SN) e estruturas do TM, como a SCP e as cpCS, ligadas à organização do comportamento relacionado ao medo e à elaboração de processos antinociceptivos, avaliando-se o envolvimento da interação entre os sistemas opioide e canabinoide e a via nigro-tectal GABAérgica na modulação do comportamento de defesa e da antinocicepção induzida pelo medo evocado pela microinjeção de antagonista GABAérgico no continuum compreendido pela coluna dorsolateral da SCP e pelas cpCS. Com esse propósito, foram estudados os efeitos de microinjeções de agonistas e de antagonistas de receptores opioides ou canabinoides não-seletivos e seletivos na substância negra, parte reticulada (SNpr), sobre a antinocicepção que segue as diversas respostas comportamentais evocadas por microinjeções de bicuculina na SCPdl/cpCS de Rattus norvegicus (Rodentia, Muridae). O presente trabalho mostrou que a microinjeção do antagonista de receptores GABAA, bicuculina no TM, induziu comportamentos defensivos, elaborados concomitantemente com processos antinociceptivos, a interação entre vias opioides e aquelas mediadas por endocanaboinoides SNpr modula o comportamento de defesa organizado no mesencéfalo dorsal sem alteração na antinocicepção induzida pelo medo. O pré-tratamento na substância negra, parte reticulada com agonistas opioides e canabinoides aumentou os limiares nociceptivos e a microinjeção de antagonistas opioides e canabinoides, causou redução dos limiares nociceptivos. Esses dados sugerem uma interação entre vias opioides e endocanabinoides da SNpr, na modulação do comportamento que tem sido relacionado ao medo inato e a ataques de pânico, sendo recrutados receptores endocanaboinoides do tipo CB1 e CB2 do mesencéfalo ventral, ao lado de receptores opioides do tipo µ1 e na modulação de vias GABAérgicas de projeção nigro-tectal. / There is a great scientific interest in searching the neuropsychopharmacological bases of behavioural reactions associated to fear and panic. Many studies suggest that the mesencephalic tegmentum (MT) a mesencephalic division rich GABA, opiod and endocannabinoid containing neurons and/or receptors complex control on defensive responses during imminent danger conditions. It is also known that the periaquedutal grey matter (PAG), the deep layers of colliculus superior (cpCS) and the colliculus inferior (CI) are important structures related to innate fear and defence as well as to the organization of fear-induced antinociception. In addition neo-striatal-nigrotectal pathways are involved in the modutation of defensive responses elaborated in the dorsal midbrain, the central mesencephalic is rich in endocannabinoids. There are interactions between opioid and GABAergic pathways in these processes. The aim of this work is to study the role of the interaction between opioid anda endocannabinoide-mediated neurotransmission on the activity of GABAergic nigro-collicular pathways. Microinjections of non-selective ande selective agonist and antagonists of opioid an canabinoid receptor were performed in the SNpr before the GABAA receptor blockade in the dorsal midbrain (SCPdl/cpCS). The GABAA receptor blockade in the Mesencephalic tectum elicited vigorous defensive behaviour. This explosive escape behaviour was followed by significant antinociception. Microinjection of opioid and cannabinoid agonists in the SNpr increased the fear-induced antinociception and the treatment of the ventral midbrain with antagonists caused opposite effect .These data suggest a clear interaction between opioids and endocannabinoids pathways of the SNpr, in the modulation of the behaviour that has been related to the innate fear and the attacks of panic, being enlisted receiving endocannabinoids of type CB1 and CB2 of mesencephalic tegmentum, to the side of opioids receptors (-opioid receptor antagonist and µ1-opioid receptor antagonist) in the modulation of nigro-tectal GABAergic pathways.

O enriquecimento ambiental (EA) é capaz de alterar a percepção a estímulos nociceptivos, bem como de aumentar a resposta analgésica induzida por opioides. Considerando que a dor neuropática é um grave problema de saúde pública e o tratamento para esta condição ainda é insatisfatório e acarreta efeitos adversos severos, os objetivos deste trabalho foram avaliar a interferência do bem-estar animal na sensibilidade dolorosa de ratos frente a diferentes estímulos nociceptivos e investigar possíveis mecanismos envolvidos neste efeito. Os animais foram submetidos à avaliação da ansiedade e da sensibilidade dolorosa, em modelo de neuropática, frente a estímulos nociceptivos mecânicos e térmicos. Foi verificado que um protocolo de EA elaborado e iniciado desde o nascimento foi capaz de reverter totalmente a dor neuropática de animais submetidos à constrição crônica do nervo isquiático (CCI). Este efeito foi completamente abolido quando os animais enriquecidos foram tratados com naloxona, um antagonista opioide não seletivo. Análises de Western Blot não mostraram diferenças na expressão de receptores opioides em regiões relacionadas ao processamento e controle da dor, porém os níveis circulantes de beta-endorfina e met-encefalina aumentaram na presença de dor crônica nos animais enriquecidos. Os níveis séricos de corticosterona também se apresentaram aumentados nos animais com EA, independentemente da neuropatia, mas o tratamento com mifepristona, um antagonista de receptores de glicocorticoides, não alterou a analgesia dos animais operados. Ainda, o EA também reduziu a imunorreatividade para serotonina na medula espinal de animais com CCI. Além do efeito analgésico, o EA também reduziu o marcador de lesão neuronal ATF-3 no gânglio da raiz dorsal e, no local da constrição, reduziu a degeneração neuronal característica do modelo, induzindo ainda, a presença predominantemente de macrófagos do tipo M2. Este trabalho reforça a importância do bem-estar na prevenção do desenvolvimento da dor neuropática e mostra uma abordagem não farmacológica que pode aumentar a resiliência de animais contra estímulos nocivos. / Environmental enrichment (EE) is capable of altering the perception of nociceptive stimuli, as well as increasing the analgesic response induced by opioids. Considering that neuropathic pain is a serious public health problem and the treatment for this condition is still unsatisfactory and induces severe side effects, the aims of this study were to evaluate the interference of animal welfare in the sensitivity to different nociceptive stimuli and to investigate possible mechanisms involved in this effect. Animals were submitted to the evaluation of anxiety and pain sensitivity in a model of neuropathic pain, against mechanical and thermal nociceptive stimuli. It was seen that an elaborated EE starting from birth was able to totally reverse the neuropathic pain of animals submitted to chronic constriction injury of the sciatic nerve (CCI). This effect was completely abolished when enriched animals were treated with naloxone, a nonselective opioid antagonist. Western blot analysis did not show differences in opioid receptor expression in regions related to pain processing and control, however, circulating levels of beta-endorphin and met-enkephalin were increased in the presence of chronic pain in enriched animals. Serum corticosterone levels were also increased in animals with EE regardless of neuropathy, but treatment with mifepristone, a nonselective glucocorticoid receptor antagonist, did not alter the analgesia of operated animals. Moreover, EE reduced serotonin immunoreactivity in the spinal cord of CCI animals. In addition to analgesic effect, EE also reduced the neuronal injury marker ATF-3 at the dorsal root ganglia and, at the site of constriction, decreased the neuronal degeneration characteristic of the model, inducing the presence of M2 macrophages subtype predominantly. This work reinforces the importance of well-being in preventing the development of neuropathic pain and shows a non-pharmacological approach that may increase animal resilience against noxious stimuli.

O câncer é a maior causa de morbidade e mortalidade em animais idosos de companhia, sendo a dor o principal sintoma relatado no homem. Objetivou-se, neste estudo, avaliar o emprego do tramadol para analgesia em cães com dor oncológica. Foram avaliados cães com câncer no período de agosto de 2004 a março de 2006, encaminhados ao Grupo de Dor do Serviço de Anestesia. Incluiu-se no estudo cães que permaneceram no mínimo 15 dias em tratamento e que, no momento de instituição da terapêutica com o tramadol, apresentaram dor igual ou maior que 4, de acordo com Escala Numérica Verbal (ENV) quando já medicados com dipirona associada ou não a antiinflamatórios não esteroidais ou esteroidais há mais de 10 (dez) dias. Avaliou-se a dor com a ENV e o escore de qualidade de vida (QV) através de escala validada para cães (Yazbek; Fantoni, 2000). Foram avaliados para o estudo 130 animais com câncer, 53 machos e 77 fêmeas, dos quais 37 obedeceram aos critérios de inclusão para análise da terapia analgésica. De acordo com a avaliação dos proprietários, a média de dor dos animais avaliados pela da ENV no momento de instituição da terapêutica com o tramadol foi de 6,11 ± 1,81 e o escore de qualidade de vida foi 21,95 ± 5,96. No primeiro retomo velificou-se melhora da dor em 31 (83,78%) animais. Neste momento foi realizada adequação da dose do tramadol, considerando-se a intensidade da dor em 20 (54,05%) dos 37 cães era superior a 4. No segundo retomo 34 (91,89%) dos animais apresentaram alívio da dor em relação à consulta inicial, sendo que 28 (75,68%) destes obtiveram ENV inferior a 4. A dose inicial preconizada do tramadol foi de 2mg/Kg a cada 8 horas, por via oral, sendo esta dose reajustada no retomo caso o valor de ENV fosse maior que 4. Classificando a eficácia do tratamento realizado com o tramadol em três categorias: alívio da dor (ENVinicial > ENVfinal e ENVfinal < 4), melhora insatisfatória (ENVinicial > ENVfinal e ENVfinal > 4) e ausência de melhora (ENVinicial &le; ENVfinal) pode-se afirmar que no primeiro retomo 83,78% e 78,28% dos pacientes, na opinião de proprietário e pesquisador respectivamente, experimentaram alguma melhora da dor. Seguindo esta mesma classificação observou-se que, no segundo retomo após acerto da dose do tramadol, o alívio da dor foi obtido em 28 (75,68%) animais na opinião de proprietário e pesquisador e melhora insatisfatória em 6 (16,21%) e 7 (18,91%) pacientes, de acordo com o proprietário e pesquisador respectivamente. Quando comparados os grupos separadamente verifica-se que o grupo DAINET (dipirona, AINE, tramadol) apresentou significante alívio da dor em relação aos primeiro retorno e a avaliação final, já os grupos DT (dipirona, tramadol) e DET (dipirona, esteróide, tramadol) apresentaram alívio da dor apenas em relação a avaliação final. Com base no alívio da dor e no aumento da qualidade de vida, pode-se concluir que o tramadol foi eficiente no tratamento da dor de grau moderada à intensa em cães portadores de câncer. / Cancer is the main cause of morbidity and mortality in companion aged dogs, being pain the principal symptom described in humans. This study aimed to evaluate the use of tramadol for analgesia in dogs with oncologic pain. Between august 2004 and marc 2006, dogs with cancer referred to the Pain Group of the Anesthesia Service were evaluated. In this study , dogs with at least fifteen days of treatment were included and that at the moment at tramadol administration, presented pain equal or above 4 according the Visual Analogue Scale (VAS) when treated with dypirona associated our not to non-esteroidal anti-inflammatory analgesics or steroidal for more than 10 days. Pain evaluated with the VAS and the quality of life (QV) score validated for dogs (Yazbek; Fantoni, 2000) were used. One hundred and thirty seven dogs with cancer were evaluated, but only 37 could be included the evaluation of analgesic therapy. According to the owner of the animals by means of the VAS at the begging of tramadol was 6,11 ± 1,81 and the QV score was 21,95 ± 5,96. At the first return, an improvement in pain was verified in 31 animals (83,78%). In this moment, adequacy of tramadol dose was performed, since the intensity of pain was above 4 in 20 (54,05%) animals. At the second returns 34 (91,89%) of the animals presented pain relief in relation to the first visit, being that 28 (75,68%) of the animal presented a VAS bellow 4 the initial dose o tramadol was 2mg/Kg each 8 hours orally, and the dose was readjusted in the return who the animal presented VAS above 4. Efficacy of treatment was classified in the three steps pain relief: successful pain relief (initial VAS > final VAS and final VAS < 4), unsuccessful pain relief (initial VAS > final VAS and final VAS > 4) and no pain relief (initial VAS &le; final VAS). It could be inferred that at the first return 83,78% and 78,28% of patients regarding owner and researcher opinion respectively obtained some pain relief. Following this some classification, it was observed that at the second return after adjusting the tramadol dosage, pain relieve was achieved in 28 (75,68%) of the animals the according to the owner and researcher and that an unsuccessful relief in 6 (16,21%) and 7 (18,91%) of patients respectively. When the groups were compared separately it was verified that the group DAINET (dypirona, AINE and tramadol) showed a significant pain relief in relation to the final evaluation, whereas the other two groups (DT - dypirona plus tramadol - and DET - dypirona, steroidal anti-inflammatory analgesic and tramadol) presented pain relief only in relation to the final evaluation. Based on the results of pain relief and QV it could be concluded that tramadol was efficient regarding the treatment of moderate to severe pain in dogs with cancer.

Orientador: Carlos Roberto Teixeira / Banca: Stelio Pacca Loureiro Luna / Banca: Lídia Mitsuko Mitsubara / Banca: Helcya Mime Hulse / Banca: Eliana Reiko Matushima / Resumo: O presente estudo aborda os efeitos causados pela contenção química com metadona (0,1mg/kg), dexmedetomidina (5µg/kg) e cetamina S(+) (8mg/kg) em macacos bugios (Alouatta guariba clamitans), bem como compara a qualidade e o tempo na reversão anestésica com antagonistas α2-adrenérgicos isolados ou associados a antagonistas narcóticos em macacos do novo mundo. Foram utilizados cinco macacos bugios, adultos e hígidos. O protocolo anestésico foi aplicado, por via intramuscular. Cinco minutos após a sedação do animal, o mesmo foi colocado em mesa com colchão térmico, onde foram aferidos os seguintes parâmetros: frequência cardíaca, respiratória, temperatura retal, pressão arterial sistólica e reflexos palpebrais. Estes parâmetros foram aferidos em intervalos de 10 minutos. Após 45 minutos da anestesia, foi aplicado a solução NaCl 0,9% para o tratamento controle, 25µg/kg de atipamezol para o tratamento ATI, 0,5 mg/kg de ioimbina para o tratamento IOI, atipamezol (25µg/kg) com naloxona (0,2 mg/kg) para o tratamento ATINALOX e o tratamento IOINALOX composto de ioimbina (0,5 mg/kg) associado à naloxona (0,2 mg/kg), administrados, por via IV, na veia ulnar dos primatas. Após a aplicação do reversor, o animal foi colocado em uma gaiola para avaliação da recuperação feita por 3 pesquisadores. O período de recuperação total foi compreendido desde a aplicação do antagonista até a total recuperação do paciente. A recuperação foi avaliada utilizando-se escores variavam de zero (0) a três (3), de acordo com Galante (2013). Alguns animais apresentaram sialorréia após a aplicação IV de atipamezol e ioimbina. Concluiu-se que a associação de metadona, dexmedetomidina e cetamina foi eficaz para contenção química desses animais, permitindo a realização de procedimentos clínicos com duração de até 45 minutos. O tratamento IOINALOX foi mais eficaz no quesito tempo, porém o tratamento ATINALOX obteve a melhor / Abstract: The present study addressed the effects caused by chemical restraint with methadone (0.1mg/kg), dexmedetomidine (5µg/kg) and ketamine S(+) (8 mg/kg) in Howley monkeys (Alouatta guariba clamitans) as well as the comparison of the quality and the time of the anesthetic reversal with isolated or associated narcotic α2-adrenergic antagonists in New world monkeys. Five adult and healthy Howley monkeys were used. The anesthetic protocol was administered via intramuscular. Five minutes after sedation, the animal was put on a table with thermal mattress where the following parameters were measured: cardiac and respiratory frequencies, rectal temperature, systolic arterial pressure and palpebral reflexes. These parameters were measured every 10 minutes. After 45 minutes of anesthesia a solution of NaCl 0.9% was administered to the control treatment, 25µg/kg of atipamezole for the ATI treatment, 0.5 mg/kg of yohimbine to the IOI treatment, atipamezole (25µg/kg) associated with naloxone (0.2 mg/kg) to the ATINALOX treatment and the IOINALOX treatment was composed of yohimbine (0.5 mg/kg) associated with naloxone (0.2 mg/kg), administered via intravenous in the primates' ulnar vein. After the reverser administration the animal was placed on a cage and the period of full recovery was evaluated by three researchers. The period of full recovery was determined from the antagonist administration until the patient's full recovery. The recovery was evaluated using scores that varied from zero (0) to three (3), accordingly to Galante, 2013. Some animals showed drooling after the intravenous administration of atipamezole and yohimbine. The association of methadone, dexmedetomidine and ketamine was effective to the chemical restraint of these animals allowing the performance of clinical procedures by up to 45 minutes. The IOINALOX treatment was the most effective regarding to time but the ATINALOX treatment gained the best recorvery / Doutor

This report describes the use of small molecule probes in the modulation of brain chemistry with the ultimate goal of developing novel therapeutics for the treatment of mood disorders. With an increasing number of people suffering from depression, there is a need to explore more diverse mechanisms of these diseases to better understand their cause and therefore provide insight into their treatment. Chapter 1 serves as an introduction and describes the current understanding of depression mechanisms, as well as a history of antidepressant therapeutics. The chapter then goes on to discuss, in depth, the mechanisms of G Protein-Coupled Receptor (GPCR) function and the implications of biased signaling. There is also an introductory overview of basic pharmacological terms. The chapter finishes with a summary of current technology available to measure GPCR function, including those utilized in the rest of this report. The remainder of the report is broken up into two parts. In the first part, I will describe my work to understand the opioid receptor system in the context of mood disorders. In Chapter 2, the atypical antidepressant tianeptine is discovered to act through the mu-opioid receptor (MOR), and a biochemical exploration is reported including an exploration of its unique properties in the context of G protein-dependent and -independent signaling, as well as preliminary in vivo and structure activity relationship studies into the mechanism of action. In Chapter 3, I will describe the biological characterization of the Mitragyna speciosa alkaloids at the opioid receptors. In particular, the major alkaloids mitragynine and 7-OH mitragynine are found to be partial agonists at the MOR and antagonists at the kappa-opioid receptor (KOR) with apparent G protein bias. In Chapter 4, alkaloids inspired by those found in Tabernanthe iboga, such as ibogaine, are synthesized and characterized at the opioid receptors. Through a novel 12- hydroxy-oxaibogamine scaffold, opioid activity is uncovered that is greatly increased in comparison to the ibogaine metabolite noribogaine. Analogs tested have varying degrees of potency and efficacy at all three opioid receptors, and one analog in particular is found to be a selective G protein biased partial KOR agonist. In Chapter 5, I will conclude the opioid section by taking a critical examination of commonly used assays for measuring arrestin recruitment by dissecting assay components and analyzing what is necessary to determine accurate calculations of bias within a cellular system. The alleged G protein bias of KOR agonist dynorphin is studied at great length, and a discussion on the future of understanding ligand bias is presented. In the second part of this report, I move away from opioids and instead focus on the growth factor signaling system as a second approach to uncovering novel therapeutics for depression. In Chapter 6, I describe a second potential mechanism of action of the natural product ibogaine in the context of glial cell line-derived neurotrophic factor (GDNF) signaling. The deconstructed iboga analog XL-008 is studied that is a superior releaser of GDNF and potentiates the signaling of a second growth factor, fibroblast growth factor 2 (FGF2). In the final Chapter 7, I look to the FGF family, both receptor and growth factor, as a novel target for depression. In order to identify small molecule modulators of the FGF receptor 1 (FGFR1), cell- based assays are developed and validated in a pilot screen. The strength of these assays are assessed, and the initial results from a full high throughput screen are presented.

A técnica de Mobilização Neural (MOB) é um método não invasivo que demonstrou tanto na pesquisa básica, como na pesquisa clinica ser eficaz na redução da sensibilidade à dor. O presente, estudo visa examinar os efeitos da MOB na disfunção locomotora, na força muscular, nas alterações morfológicas no nervo isquiático e nas alterações moleculares induzida pela constrição crônica (CCI) do nervo isquiático de ratos Wistar. Para analisar a disfunção locomotora utilizamos o índice funcional do nervo Isquiático (IFC). Para analisar a força muscular, o sistema Biopac System. A ultraestrutura do nervo foi analisada pela técnica de microscopia eletrônica de transmissão e as alterações moleculares por meio de ensaios de Western blot. Ao finalizarmos os tratamentos com MOB os animais foram eutanasiados e os tecidos como, nervo isquiático, gânglios das raizes posteriores (DRG L4-L6) e Susbtância Cinzenta Periaquedutal (PAG) foram retirados. Os DRG´s foram processados pela técnica de Western Blot para a detecção da substância P (SP), receptor de potencial transitório vanilóide tipo I (TRPV1) e receptores opióides dos tipos &#181; (MOR), &delta; (DOR) e k (KOR). Com relação a PAG, analisamos somente os receptores opióides por Western Blot. Nossos resultados demonstraram uma reverção da disfunção locomotora induzida pela CCI após a MOB e aumentou 172% a força do músculo tibial anterior nos animais tratados quando comparado com os animais do grupo CCI. Nossos estudos sobre a ultraestrutura do nervo isquiático demonstraram intenso processo de degeneração Waleriana após a CCI e regeneração após a MOB. Podemos sugerir um papel importante da MOB na modulação da expressão da SP e do TRPV 1. Sobre os receptores DOR e KOR no DRG, não encontramos alterações estatísticas entre os grupos, mas observamos um aumento da expressão de MOR após a MOB. Na PAG, nós observamos uma diminuição de DOR e KOR no grupo CCI e aumento após a MOB. Por outro lado, não encontramos alterações estatíticas para o receptor MOR. Baseado nestes achados, podemos sugerir que a MOB reverte a disfunção locomotora, aumenta a força muscular, induz a regeneração do nervo isquiático, modula a SP e TRPV 1 e aumentou a expressão de MOR no DRG´s. Sugerimos ainda que, a analsegia induzida pela técnica de MOB possa ter um envolvimento também com o sistema inibitório descendente de dor resultando na inibição da transmissão do estímulo nociceptivo aferente e assim, diminuindo a dor neuropática devido influência da MOB sobre os opióides na PAG. / Neural mobilization technique (MOB) is a noninvasive method that demonstrated to be effective in reducing pain sensitivity in both clinical and research study. The present study aims to examine the effects of MOB in locomotors dysfunction, muscle strength, morphological changes in sciatic nerve and molecular changes induced by chronic constriction (CCI) of the sciatic nerve in Wistar rats. To analyze locomotors dysfunction we used the Sciatic nerve functional index (SFI). To analyze muscle strength, was used Biopac System. The nerve morphology was analyzed using electron microscopy and molecular changes through western blot assays. After MOB treatments, animals were euthanized and tissues such as, sciatic nerve, the posterior root ganglions (DRG L4-L6) and substance periaqueductal gray (PAG) were removed. The DRG were processed by western blot for detection of substance P (SP), transient receptor potential vanilloid type I (TRPV1) and opioids receptors (MOR, DOR, KOR). Regarding PAG, we analyze only opioids receptors. Our results demonstrated a full reversal of locomotors dysfunction-induced by CCI after MOB treatment and an increase of 172% on maximal tetanic muscle strength in animals treated with MOB when compared to the CCI group. Our studies on photomicrography of sciatic nerve showed an intense Wallerian degeneration process in CCI animals and an intense regeneration of myelinated fibers. In western blot assays, we identified, in DRG, an increase of SP and TRPV1 expression after CCI and a decrease of optical density after MOB treatment. Regarding opioid receptor, we did not identify statistical changes on DOR and KOR in DRG, but we observed an increased expression of MOR in CCI after MOB treatment group. In PAG analyses, we observed a decrease in DOR and KOR expression after MOB treatment when compare with CCI animals. On the other hand, we did not identify any changes on MOR receptor. Based on our findings, we suggest that treatment with neural mobilization technique it is able to reverses the locomotors dysfunction and increases maximum tetanic force of the tibialis anterior muscle after CCI. Furthermore, the same treatment was also able to induce a severe regeneration in the sciatic nerve after treatment. Still, we can suggest an important role of MOB in modulating SP and TRPV 1 expression. We suggest that antinociceptive effect induced by MOB technique can also be involved with descending pain inhibitory system resulting in inhibition of the transmission of afferent nociceptive stimulus and thereby reducing neuropathic pain because of the influence of MOB opioids in the PAG.

Made available in DSpace on 2016-04-28T20:18:31Z (GMT). No. of bitstreams: 1 2008 - Cristiane Belchior Caloiero.pdf: 424554 bytes, checksum: 418546c1b267e0fbd474d742aa991711 (MD5) Previous issue date: 2008-05-08 / The ovariohysterectomy (OHE) is the most common surgery realized in the small animals hospitals in order to control animal population and to prevent future problems of reproductive order. This surgical procedure has a moderate degree of pain and requires analgesic treatment. Postoperative pain, when not correctly diagnosed and treated, instead of being a mechanism of natural defense, becomes harmful and promote systemic alterations that delays the recovery of the patient. The objective of this study was to compare the analgesic effect in the trans and postoperative period between morphine, tramadol and buprenorphine, that were given to cats prior having OHE. In this study, 30 cats were used, with no specific breed and healthy. The animals were distributed in three groups of 10 animals. Group M received daily pre-anesthesic medication (PAM), acepromazine maleate (0.05 mg/kg) and morphine sulfate (0.2 mg/kg) intramuscularly (IM). Group T received acepromazine (0.05 mg/kg) and Tramadol (2.0 mg/kg) IM and Group B received acepromazine in the same dose and buprenorphine (0.01 mg/Kg) IM. After 20 min of applying PAM, the animals received an induction of the anesthesic thiopental 2.5% in the dose of 12.5 mg/kg intravenously. The animals were kept with isoflurane diluted in 100% oxygen through the universal vaporizer, connected to a circuit of Mapleson D (Baraka). The evaluation of pain and sedation was done by one person who observed for all the animals, and he did not know the treatment used (double blind). The evaluation times were: daily presurgical; 10 min, 20 min and 30 min of surgery and 1, 2, 4 and 6 hours after surgery. The subjective evaluation of pain was done through a visual analogue scales (VAS) and through a descriptive scale before and after the surgery. The measurements of cardiac frequency (FC), respiratory frequency (FR), temperature (Temp), plasma glucose (GL), time of capillary perfusion (TPC) and analysis of mucosa were made moments before, during, and after the surgery. The gauging of the systolic arterial pressure (PAS) and saturation of oxygen hemoglobin (SpO2) were made only during the surgical procedure. The comparison of the objective and subjective parameters between the three groups was done by using the Analysis of Variance of Kruskal-Wallis, followed of the Mann-Whitney test, whenever were significant differences between the treatments (p< 0.05). Inside of each group, the Analysis of Variance of Friedman was used for the comparisons between the diverse moments, followed by the test of Wilcoxon for the identification of the different moments (p< 0.05). Animals of the buprenorfina group (B) were more sedate in all the evaluated moments, followed by the group morphine (M) and finally the group tramadol (T). In the evaluation of pain for the VAS, the group tramadol demonstrated a lesser degree of pain in the first the 2 hours, followed by the groups with morphine and buprenorphine. In the evaluation of pain for the descriptive scale, tramadol presented better analgesic effect at the times 2, 4, and 6 hours. The values of cardiac frequency remained relatively constant during the surgical procedure, only being different at the times 4 and 6 hours where the buprenorphine group presented higher values. In the present study, the reduction of the FR was more accentuated in the morphine group. Difference in the PAS between the groups was not observed. The buprenorphine group presented higher values of glucose at the moment 6 hours. In this study, the body temperature did not differ between the groups in the pre or postoperative moments. With this study, it can be concluded that tramadol, in the given dose and regimen, is more efficient than the morphine and buprenorphine in controlling of postoperative pain of OHE in cats, without signs of collateral effect. / A ovariossalpingo-histerectomia (OSH) ? a cirurgia mais comumente realizada na cl?nica de pequenos animais com objetivos de controlar a popula??o e evitar problemas futuros de ordem reprodutiva. Este procedimento cir?rgico cursa num grau de dor moderado requisitando de um tratamento analg?sico adequado. A dor p?s-operat?ria, quando n?o corretamente diagnosticada e tratada, deixa de ser um mecanismo de defesa natural e torna-se nociva, promovendo altera??es sist?micas relevantes que retardam a recupera??o do paciente. O objetivo deste trabalho foi comparar o efeito analg?sico no per?odo trans e p?s-operat?rio entre morfina, tramadol e buprenorfina administrados preventivamente em gatas submetidas a OSH. Neste estudo foram utilizadas 30 gatas, sem ra?a definida e h?gidas. Os animais foram distribu?dos aleatoriamente em tr?s grupos de 10 animais. O grupo M recebeu de medica??o pr?-anest?sica (MPA), maleato de acepromazina (0,05 mg/kg) e sulfato de morfina (0,2 mg/kg) pela via intramuscular (IM). O grupo T recebeu acepromazina (0,05 mg/kg) e Tramadol (2 mg/kg) pela via IM. Ao grupo B foi administrado acepromazina na mesma dose e buprenorfina (0,01 mg/Kg) via IM. Ap?s 20 min da MPA, os animais recebiam de indu??o anest?sica tiopental 2,5% na dose de 12,5 mg/kg pela via intravenosa. Os animais eram intubados e mantidos com isoflurano dilu?do em oxig?nio a 100% atrav?s do vaporizador universal, conectados a um circuito de Mapleson D (Baraka). A avalia??o da dor e seda??o foi realizada por um mesmo observador em todos os animais, e este n?o conhecia o tratamento utilizado (duplo cego). Os tempos de avalia??o eram: pr?-operat?rio; 10, 20 e 30 minutos de cirurgia, 1, 2, 4 e 6 horas ap?s o t?rmino da mesma. A avalia??o subjetiva da dor foi realizada atrav?s da escala an?loga visual e da escala descritiva nos momentos antes e ap?s a cirurgia. A mensura??o dos par?metros objetivos freq??ncia card?aca (FC), freq??ncia respirat?ria (FR), temperatura (Temp), glicemia (GL), tempo de perfus?o capilar (TPC) e an?lise de mucosas foram realizados nos momentos antes, durante e ap?s a cirurgia, j? a aferi??o da press?o arterial sist?lica (PAS) e satura??o de oxig?nio na hemoglobina (SpO2) foram realizadas somente durante o procedimento cir?rgico. A compara??o dos par?metros objetivos e subjetivos entre os tr?s grupos foi realizada pela An?lise de Vari?ncia de Kruskal-Wallis, seguido do teste Mann-Whitney, sempre que houvesse diferen?as significativas entre os tratamentos (p< 0,05). Dentro de cada grupo, foi utilizada pela An?lise de Vari?ncia de Friedman para as compara??es entre os diversos momentos, seguido do teste de Wilcoxon para a identifica??o dos momentos diferentes (p< 0,05). Os animais do grupo buprenorfina (B) se apresentaram mais sedados em todos os momentos avaliados, seguido do grupo morfina (M) e por ?ltimo o grupo tramadol (T). Na avalia??o da dor pela VAS, o grupo tramadol demonstrou um menor grau de dor nas primeiras 2 horas, seguido pelos grupos morfina e buprenorfina. Na avalia??o da dor pela escala descritiva, o tramadol apresentou melhor efeito analg?sico nos momentos 2, 4, e 6 horas. Os valores de freq??ncia card?aca permaneceu relativamente constante durante todo o procedimento cir?rgico, s? sendo diferente nos momentos 4 e 6 horas onde a buprenorfina apresentou valores mais altos. No presente estudo, a diminui??o da FR foi mais pronunciada no grupo morfina. N?o foi observada diferen?a na PAS entre os grupos. O grupo buprenorfina apresentou valores mais altos de glicose no momento 6 horas. Neste estudo, a temperatura corporal n?o diferiu entre os grupos nos momentos pr? e p?s-operat?rio. Com este estudo, pode-se concluir que o tramadol na dose e regime empregados mostrou-se mais eficiente que a morfina e a buprenorfina em controlar a dor p?s-operat?ria da OSH em gatas, sem apresentar efeitos colaterais.

Existe um grande interesse científico voltado para a busca das bases neuropsicofarmacológicas dos comportamentos que têm sido associados ao medo e ao pânico. Muitos estudos sugerem o teto mesencefálico (TM) como responsável pelo controle de respostas defensivas elaboradas durante situações de perigo iminente. A substância cinzenta periaquedutal (SCP), as camadas profundas do colículo superior (cpCS) e o colículo inferior (CI) têm sido considerados importantes estruturas na elaboração do medo inato e do comportamento de defesa, assim como na organização da antinocicepção induzida pelo medo inato. Contudo, muitos estudos têm implicado a via neoestriado-nigro-tectal no controle de respostas defensivas elaboradas no mesencéfalo dorsal, permeadas pela interação entre vias opioides e GABAérgicas. O presente trabalho tem por objetivo o estudo neurofarmacológico da anatomia conectiva funcional entre a substância negra (SN) e estruturas do TM, como a SCP e as cpCS, ligadas à organização do comportamento relacionado ao medo e à elaboração de processos antinociceptivos, avaliando-se o envolvimento da interação entre os sistemas opioide e canabinoide e a via nigro-tectal GABAérgica na modulação do comportamento de defesa e da antinocicepção induzida pelo medo evocado pela microinjeção de antagonista GABAérgico no continuum compreendido pela coluna dorsolateral da SCP e pelas cpCS. Com esse propósito, foram estudados os efeitos de microinjeções de agonistas e de antagonistas de receptores opioides ou canabinoides não-seletivos e seletivos na substância negra, parte reticulada (SNpr), sobre a antinocicepção que segue as diversas respostas comportamentais evocadas por microinjeções de bicuculina na SCPdl/cpCS de Rattus norvegicus (Rodentia, Muridae). O presente trabalho mostrou que a microinjeção do antagonista de receptores GABAA, bicuculina no TM, induziu comportamentos defensivos, elaborados concomitantemente com processos antinociceptivos, a interação entre vias opioides e aquelas mediadas por endocanaboinoides SNpr modula o comportamento de defesa organizado no mesencéfalo dorsal sem alteração na antinocicepção induzida pelo medo. O pré-tratamento na substância negra, parte reticulada com agonistas opioides e canabinoides aumentou os limiares nociceptivos e a microinjeção de antagonistas opioides e canabinoides, causou redução dos limiares nociceptivos. Esses dados sugerem uma interação entre vias opioides e endocanabinoides da SNpr, na modulação do comportamento que tem sido relacionado ao medo inato e a ataques de pânico, sendo recrutados receptores endocanaboinoides do tipo CB1 e CB2 do mesencéfalo ventral, ao lado de receptores opioides do tipo µ1 e na modulação de vias GABAérgicas de projeção nigro-tectal. / There is a great scientific interest in searching the neuropsychopharmacological bases of behavioural reactions associated to fear and panic. Many studies suggest that the mesencephalic tegmentum (MT) a mesencephalic division rich GABA, opiod and endocannabinoid containing neurons and/or receptors complex control on defensive responses during imminent danger conditions. It is also known that the periaquedutal grey matter (PAG), the deep layers of colliculus superior (cpCS) and the colliculus inferior (CI) are important structures related to innate fear and defence as well as to the organization of fear-induced antinociception. In addition neo-striatal-nigrotectal pathways are involved in the modutation of defensive responses elaborated in the dorsal midbrain, the central mesencephalic is rich in endocannabinoids. There are interactions between opioid and GABAergic pathways in these processes. The aim of this work is to study the role of the interaction between opioid anda endocannabinoide-mediated neurotransmission on the activity of GABAergic nigro-collicular pathways. Microinjections of non-selective ande selective agonist and antagonists of opioid an canabinoid receptor were performed in the SNpr before the GABAA receptor blockade in the dorsal midbrain (SCPdl/cpCS). The GABAA receptor blockade in the Mesencephalic tectum elicited vigorous defensive behaviour. This explosive escape behaviour was followed by significant antinociception. Microinjection of opioid and cannabinoid agonists in the SNpr increased the fear-induced antinociception and the treatment of the ventral midbrain with antagonists caused opposite effect .These data suggest a clear interaction between opioids and endocannabinoids pathways of the SNpr, in the modulation of the behaviour that has been related to the innate fear and the attacks of panic, being enlisted receiving endocannabinoids of type CB1 and CB2 of mesencephalic tegmentum, to the side of opioids receptors (-opioid receptor antagonist and µ1-opioid receptor antagonist) in the modulation of nigro-tectal GABAergic pathways.