Fast excitatory neurotransmission is critical for learning and memory, and its dysregulation is linked to numerous neurological diseases. These include developmental diseases such as fragile X syndrome, psychiatric disorders like schizophrenia, and chronic neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Throughout the central nervous system, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors (AMPARs) mediate the fastest excitatory neurotransmission. In response to the neurotransmitter glutamate, AMPARs open their ion channels and allow cation flux through the post-synaptic membrane. This initiates rapid depolarization and signaling in the post-synaptic neuron. Nearly all AMPARs exist as complexes with auxiliary subunits, which are regulatory proteins that modulate receptor assembly, trafficking, pharmacology and function. These auxiliary subunits determine brain region-specific AMPAR signaling, and aberrancies in complex formation or function lead to neuropathologies. Despite their importance for CNS signaling and implication in neurologic disorders, the structural bases underlying the function of AMPARs and AMPAR complexes remain ambiguous, representing a critical barrier to our understanding of excitatory neurotransmission. As a consequence, structure-based design of neuro-therapeutics is largely undeveloped: there is only a single FDA-approved drug targeting AMPARs. To address these problems, I wanted to dedicate my thesis work to study AMPAR synaptic complexes across an array of functional states and provide a new foundation for our structural understanding of AMPAR signaling. First, I designed a covalent-fusion construct approach to guarantee assembly and expression of AMPAR synaptic complexes in heterologous cells (HEK293). Then, I developed purification protocols allowing me to obtain chemically homogenous and pure complex protein. Since synaptic signaling is highly dynamic, complexes of AMPARs with auxiliary subunits are conformationally heterogeneous and are not amenable to X-ray crystallography. Cryo-electron microscopy (cryo-EM) enabled me to approach these complexes structurally, where I could collect data and parse out heterogeneity through image classification. With cryo-EM, I solved the structure of an AMPAR bound to the auxiliary subunit stargazin, which promotes AMPAR activation. This work provided the first structural information on how AMPARs form complexes with regulatory subunits. In a following study, I solved the structure of an AMPAR in complex with a functionally distinct auxiliary subunit, GSG1L. In contrast to stargazin, GSG1L promotes inactivation and desensitization of AMPARs, thus having a neuroprotective effect. To further characterize the function of these auxiliary subunits, I designed chimeras between stargazin and GSG1L and examined their function electrophysiologically. This experiment revealed that AMPAR auxiliary subunits have a modular design, where variable extracellular domain regions, supported by a conserved transmembrane α-helical bundle, distinctly regulate function of the core AMPAR. This study provided the first evidence of how brain region-specific expression patterns of similarly-structured auxiliary subunits may contribute to unique AMPAR functions. More recently, I’ve taken advantage of the modulatory effects of stargazin on AMPARs and I applied cryo-EM to an AMPAR-stargazin complex. This study determined how AMPARs are activated by the neurotransmitter glutamate, and revealed a novel mechanism by which glutamate binding induces opening of AMPAR ion channels. Our data show that two-fold symmetric kinking of ion channel helices allows cation flux into neurons, which triggers neurotransmission. Importantly, this study also provides insights into how mRNA editing and patient-derived disease mutations in the transmembrane (i.e., resulting in aberrantly firing of receptors during epilepsy) reshape AMPAR function and excitatory neurotransmission. Collectively, the findings from my thesis work provide a new paradigm for the molecular-level understanding of glutamatergic neurotransmission throughout the CNS. These studies lay the groundwork for new directions in precision-medicine design of therapeutics targeting brain region-specific AMPAR synaptic complexes in neurological diseases.

Magister Scientiae - MSc / Background: Early postnatal radiation exposure from environmental, diagnostic or therapeutic sources is potentially deleterious to the developing nervous system resulting in oxidative stress, structural damage, altered neurochemistry, DNA damage, inflammatory stresses as well as correlating cognitive impairment during adult life. Numerous studies in literature have investigated the radioprotective effects of medicinal plants and beverages. However, only a few studies have focused on the radioprotective effects of rooibos, an indigenous South African herbal tea, well known for its many acclaimed health benefits. Aims: This study was done to investigate the diverse radioprotective potential of fermented Rooibos herbal tea (FRHT) consumed ad libitum by pregnant rats on the adult offspring rats exposed to a once-off 6 Gy dose of gamma irradiation on postnatal day 3. Methods: Twenty-four (24) adult female rats were equally divided into four groups (6 per group) as control (NS), radiation (X), tea (RT) and their combination. On PND 30, offspring rats were subjected to neurobehavioural assessment for open field and novel object recognition parameters and later sacrificed, the brain tissues removed and processed for histological, immunohistochemical and neurochemical analyses, using standard techniques. Results: Pre-treatment with FRHT showed overall protection against radiation-induced distortions in offspring rats by significantly improving exploratory activity, the frequency of central square entry, rearing episodes, cumulative freezing time and memory retention as indicated by a relatively higher recognition index. FRHT was also found to significantly improve the antioxidant defence mechanisms in the offspring rats by reversing lowered FRAP levels, increasing superoxide dismutase and catalase enzyme activities and reducing lipid peroxidation. Histological and immunohistochemical analyses showed that morphological alterations were generally attenuated in the RTX group and the high number of caspase-3 and Glial fibrillary acidic protein (GFAP)-positive cells was significantly reduced, indicating protective effects against apoptosis and gliosis. Conclusion: Taken together, our findings tend to suggest that the potential radioprotective effects of FRHT are multimodal, possibly executed through the anti-apoptotic, antioxidative, anti-gliosis and other mechanisms, as observed in this study, and this is often attributed to the high polyphenol content in Rooibos tea.

Orientador: Ana Marisa Fusco Almeida / Coorientador: Jean Leandro dos Santos / Coorientador: Maria José Soares Mendes Giannini / Banca: Daniel Assis dos Santos / Banca: Luciana Trilles / Banca: Valéria Valente / Banca: Reginaldo dos Santos Pedroso / Resumo: Criptococose é uma importante micose sistêmica que acomete principalmente pacientes imunocomprometidos e é classificada como a infecção fúngica com maior mortalidade entre indivíduos portadores de HIV. Cryptococcus neoformans é uma levedura capsulada e o principal agente etiológico da criptococose; encontra-se dispersa no meio ambiente na forma de basidiósporos, os quais são responsáveis pela infecção em humanos e animais. As principais manifestações clínicas estão associadas à infecção pulmonar e meningite. A terapia se dá basicamente com a administração de anfotericina B (AMB) na terapia de ataque associada ou não a 5-fluocitosina e como manutenção é indicado ao longo do tratamento o fluconazol (FCZ). Apesar de esta terapêutica ser eficiente, é constatado elevado número de casos de reincidência e desenvolvimento de resistência aos azóis, além de problemas de toxicidade. Diante desta problemática, este estudo propõe o desenvolvimento de novas alternativas terapêuticas para o tratamento da criptococose, visando duas abordagens distintas. A primeira, aplicando novo composto antifúngico e a segunda, o estudo de microRNAs (miRNAs) envolvidos na interação da levedura e células de glioblastoma humano (U87-MG), com a finalidade de usá-los como reguladores da infecção, sendo esta uma estratégia recentemente divulgada em uma variedade de doenças. Para tanto, inicialmente moléculas análogas de resveratrol foram avaliadas quanto a atividade antifúngica e o derivado orto,orto-HDZ (HDZ) mostrou forte atividade fungicida contra isolados de C. neoformans, com valores de concentração fungicida mínima (CFM) variando entre 0,97 a 7,81 µg/mL. A associação entre HDZ e FCZ mostrou sinergia com potencialização do efeito do FCZ em até 64 vezes para um isolado clínico resistente. Baixa toxicidade foi... / Abstract: Cryptococcosis is an important systemic mycosis that mainly affects immunocompromised patients and is classified as the fungal infection with higher mortality among individuals with HIV. Cryptococcus neoformans is an encapsulated yeast and the main etiologic agent of cryptococcosis. This yeast is dispersed into the environment in the form of basidiospores, which are responsible for infection in humans and animals. The main clinical manifestations are associated with pulmonary infection and meningitis. The treatment is basically the administration of amphotericin B (AMB) as therapy consolidation with or without the use of 5-flucytosine and, for the maintenance therapy, fluconazole (FCZ) is indicated. Although this therapy is effective, it is observed high number of cases of relapses, development of resistance to azoles and toxicity problems. In front of these problems, this study proposes the development of new therapies for the treatment of cryptococcosis, targeting two distinct approaches. The first aims a new antifungal compound and the second the study microRNAs (miRNAs) involved in the interaction between yeast and human glioblastoma cells (U87-MG), in order to use them as regulators of infection, since this is a recently disclosed strategy in a variety of diseases. For this purpose, initially, analogs of resveratrol molecule were evaluated for antifungal activity and the derived ortho,ortho-HDZ (HDZ) showed a strong fungicidal activity against isolates of C. neoformans with values of minimum fungicidal concentration (MFC) ranging between 0.97 to 7.81 µg/mL. The association between HDZ and FCZ showed synergy with potentiation of the effect of FCZ in up to 64 times for a resistant clinical isolate to FCZ. Low toxicity was observed in in vitro and in vivo assays (Galleria mellonella and mice), despite showing changes in ... / Doutor

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A cumarina (1,2-benzopirona) Ã um composto aromÃtico encontrado em vÃrias espÃcies vegetais. Este trabalho objetivou avaliar as aÃÃes da cumarina em modelos comportamentais de ansiedade, depressÃo e sedaÃÃo, tais como, campo aberto, rota rod, labirinto em cruz elevado (LCE), placa perfurada e suspensÃo da cauda, procurando esclarecer os mecanismos atravÃs de doseamento de aminoÃcidos em cÃrtex prÃ-frontal e hipocampo de camundongos atravÃs de HPLC (High Perfomance Liquid Chomatography). Foram utilizados camundongos albinos, variedade Swiss Webster, adultos, machos, pesando entre 25-30 g, provenientes do BiotÃrio do Departamento de Fisiologia e Farmacologia da UFC. A cumarina (CUM) foi administrada de forma aguda por via intraperitoneal nas doses de 5, 20 ou 40 mg/kg. Os resultados mostram que a CUM, em todas as doses utilizadas, diminuiu a atividade locomotora, o nÃmero de rearing e grooming no teste do campo aberto, sugerindo uma possÃvel aÃÃo sedativa. No rota rod nÃo alterou a coordenaÃÃo motora ou causou dÃficit muscular nos animais, sugerindo que seus efeitos nÃo se devem ao bloqueio neuromuscular perifÃrico. No LCE e no teste da placa perfurada, a cumarina mostrou seu efeito ansiogÃnico, pois reduziu todos os parÃmetros analisados no LCE, como o nÃmero de entradas nos braÃos abertos (NEBA), percentagem de entrada nos braÃos abertos (PEBA), Tempo de permanÃncia nos braÃos abertos (TPBA) e percentagem do tempo de permanÃncia nos braÃos abertos (PTBA), assim como o nÃmero de head dips na placa perfurada. Ainda com o intuito de esclarecer as alteraÃÃes ocorridas na atividade locomotora dos animais, foi administrada a levodopa + carbidopa (L-DOPA), que levou a um pequeno aumento na atividade locomotora, em relaÃÃo ao grupo da CUM. Quando associada ao haloperidol (HALO) - antagonista dopaminÃrgico, o efeito da L-DOPA foi revertido. HALO + CUM causou uma maior interferÃncia na locomoÃÃo, como um efeito sinÃrgico. A cumarina nÃo apresentou efeito antidepressivo no teste da suspensÃo da cauda, pois aumentou o tempo de imobilidade dos animais. A imipramina (antidepressivo) diminuiu este parÃmetro. No doseamento de aminoÃcidos neurotransmissores houve aumento nos nÃveis de GABA no cÃrtex prÃ-frontal, de maneira semelhante ao diazepam, podendo explicar em parte a diminuiÃÃo da atividade locomotora. TambÃm aumentaram glutamato, glicina e taurina no grupo tratado com 20 mg/kg de cumarina. No hipocampo os nÃveis de glutamato foram significativamente reduzidos. Estas aÃÃes podem estar ligadas ao aumento ou diminuiÃÃo nos nÃveis de aminoÃcidos excitatÃrios e inibitÃrios e envolvimento dopaminÃrgico. O efeito ansiogÃnico da cumarina parece envolver a participaÃÃo da dopamina sobre o estriado. O aumento dos nÃveis de taurina leva a um balanÃo nos nÃveis de glutamato, o que pode explicar o efeito ansiogÃnico e possivelmente neuroprotetor da cumarina. A cumarina demonstrou aÃÃo sedativa e ansiogÃnica sobre o SNC provavelmente devido atuaÃÃo como antagonista dopaminÃrgico. Esse mecanismo pode ter sido modulado pelos sistemas gabaÃrgico e, principalmente, glutamatÃrgico no cÃrtex prÃ-frontal. / Coumarin (1,2-benzopyrone) is an aromatic compound found in many plant species. This study proposed to evaluate the coumarin actions in behavioral models of anxiety, depression and sedation activity, such as the open field, rota rod, elevated plus maze (EPM), hole board and tail suspension, still looking to clarify mechanisms by which this compound acts, through amino acids determination studies in mice prefrontal cortex and hippocampus by HPLC (High Performance Liquid Chomatography). The coumarin (CUM) was administered acutely in all tests, at doses of 5, 20 or 40 mg/kg, by intraperitoneal route. The results show that CUM, in all used doses, decreased the locomotor activity, the number of rearing and grooming in open field test, suggesting a possible sedative action. In the rota rod the coumarin did not alter the motor coordination or caused muscular deficit in animals. This suggests that the depressant effect of coumarin should not be exercised by peripheral neuromuscular blockade, but possibly the effects must involve neurons that control the central depressive activity. In the EPM and hole board test, coumarin proved its anxiogenic effect, as it reduced all examined parameters in the EPM, as NEOA, PEOA, TPOA and PTOA, as well as the number of head dips in the hole board. This opposite effect to diazepam was not reversed by flumazenil (benzodiazepine antagonist). This suggests that coumarin does not act in a similar manner to benzodiazepines. Still aiming to clarify the changes in animals locomotor activity, it was administered levodope + carbidope (L-DOPA), which led to a small increase in the locomotor activity, compared to CUM group. When associated to haloperidol (HALO) - dopaminergic antagonist, L-DOPA had its effects reversed. HALO + CUM caused greater interference in locomotion, as a synergic effect. The coumarin did not show antidepressant effect in the tail suspension test, because it increased the immobility time of animals. Imipramine (antidepressant) decreased this parameter. In the determination of amino acids neurotransmitters it was observed an increase in the GABA levels in the prefrontal cortex, similar to diazepam, which may partly explain the decrease in locomotor activity. The levels of glutamate, glycine and taurine also increased in the group treated with coumarin 20 mg/kg. In the hippocampus, significant changes occurred in levels of glutamate, which were reduced. It follows, therefore, that the CUM has sedative, anxiogenic and depressant activities. These actions can be linked to an increase or decrease of excitatory and inhibitory amino acids levels and dopaminergic involvement. The anxiogenic effect of coumarin seems to involve the dopamine participation on striatum. Increased levels of taurine leads to a balance in the glutamate levels, which may explain the anxiogenic effect and possible neuroprotective of coumarin. The coumarin demonstrate sedative and anxiogenic action in CNS probably caused by dopaminergic antagonism modulated by gabaergic system, mainly glutamatergic in the prefrontal cortex.

A raiva é uma doença do sistema nervoso central que é quase invariavelmente fatal. Apesar de causar cerca de 60.000 mortes/ano, a raiva ainda permanece uma doença negligenciada na maioria dos países, principalmente naqueles em desenvolvimento. O objetivo do nosso estudo foi verificar nos microambientes meningeal, perivascular e intraparenquimatoso do sistema nervoso central, o processo inflamatório, a resposta imune do hospedeiro e a morte dos neurônios frente à infecção rábica transmitida por morcegos. Verificamos que a raiva humana transmitida por morcegos é uma meningoencefalomielite. Através de reação imuno-histoquímica caracterizamos e quantificamos in situ a distribuição do antígeno viral, o fenótipo de células inflamatórias, as células expressando citocinas pró inflamatórias, citocinas representativas de padrão Th1 e Th2 e células em apoptose. O antígeno viral foi encontrado difusamente no parênquima cerebral, em maior abundância em neurônios, não diferindo sua distribuição em relação as regiões cerebrais. As células da glia, em especial os astrócitos, estavam imunomarcadas com o antígeno da raiva, assim como as células endoteliais e células mononucleadas da luz vascular. Esses achados contribuíram para a hipótese da ocorrência de uma via hematogênica alternativa de disseminação viral, através da infecção de células endoteliais pelo vírus, posterior infecção de astrócitos e finalmente infecção de neurônios. A expressão significativa de IL-12 nos pacientes rábicos provavelmente traduz imunidade de base preservada. Identificamos, outrossim, falta de resposta efetiva das células NK e comprometimento da resposta imune adaptativa seqüencial, demonstrada pela depleção de linfócitos TCD4+ no parênquima cerebral, prejuízo da atividade citotóxica dos linfócitos TCD8+ com proporcionalmente baixa expressão de granzima e expressão rarefeita de IFN-g e IL-2r. Essa situação deve ser reflexo da manipulação do vírus sobre a resposta imune inata e adaptativa do hospedeiro tendo como conseqüência uma reposta ineficaz para clareamento do vírus. A expressão aumentada de citocinas pró-inflamatórias (IL-1b, IL-6 e TNF-a) contribuiu para o dano neuronal. O padrão citocínico predominante no sistema nervoso central na raiva foi o Th2, com alta expressão de IL-4 e IL-10. O TGF-b aumentado nos casos de raiva favorece um ambiente imunossupressor para manter intacta a rede neuronal, mas também contribui para permanência local do vírus. A verificação de grau pouco acentuado de neurônios apoptóticos em contraposição a apoptose expressiva de linfócitos TCD4+ e TCD8+, indicaria a utilização pelo vírus de mecanismos ora anti-apoptóticos para preservar neurônios e favorecer a disseminação viral, ora pró-apoptóticos, destruindo linfócitos e atenuando o processo inflamatório. Em síntese, o envolvimento do sistema nervoso central em decorrência da raiva se faz às custas de processo inflamatório com predomínio local de linfócitos TCD8+, um padrão Th2 de expressão de citocinas, acometendo os microambientes meningeal, perivascular e intraparenquimatoso, sendo o bulbo a região mais afetada pelo processo. / Viral disease of central nervous system almost invariable fatal, rabies causes about 60.000 deaths yearly, and still remain a neglected disease in most countries, specially in developing ones. We study the meningeal, perivascular and parenquimal environment in central nervous system from patients who dies after bat transmitted rabies, looking for the inflammatory process, host immune response and neuronal death. We show that human rabies transmitted by bats is a meningoencephalomyelitis. Immunohistochemistry allows the in situ quantification of viral antigen distribution, inflammatory cellular phenotype, expression of cytokines representing both Th1 and Th2 profile and pro-inflammatory and cell apoptosis. Viral antigen was found disseminatted in cerebral parenquima, more abundant in neurons, without cerebral area preference. Glial cells, specially astrocytes, were immunostained with rabies antigen, as well as endothelial and vascular mononuclear cells. These findings contributed for an alternative hypothesis of the occurrence of hematogenic viral dissemination, through viral infection or passage by endothelial cells, followed by astrocyte infection, and finally reaching neurons. IL-12 significant expression in central nervous system from rabies patients probably reflect preservation of immunity. The lack of effective NK cells could compromise adaptive immune response, demonstred by TCD4+ lymphocytes depletion in cerebral parenquima, ineffective TCD8+ cytotoxic activity with low granzyme expression and low expression of IFN-g and IL-2r. This situation reflects viral effect on host innate and adaptive immune response leading to an ineffective response for viral clearance. High pro-inflammatory cytokine expression, IL- 1b, IL-6 and TNF-a, contribute for neuronal damage, with predominant Th2 cytokine profile in central nervous system in rabies patients, with high expression of IL-4 and IL-10. Increased TGF-b expression also shows an immune suppressor environment, which maintains the neuronal network intact, but also contributes for viral permanence. The low degree of apoptotic neurons opposed with expressive apoptosis of TCD4+ and TCD8+ lymphocytes, could indicate viral mechanisms anti-apoptotic for neurons preservation and favour viral dissemination, or pro-apoptotic, destroying lymphocytes and attenuating the inflammatory process. In synthesis, the involvement of central nervous system in rabies is consequence of inflammatory process with TCD8+ lymphocytes predominance, Th2 profile cytokines expression, affecting all brain compartments and areas, especially the medulla oblongata.

As enfermidades neurológicas, notadamente os tumores intracranianos, têm grande importância dentre os quadros mórbidos que acometem animais da espécie canina, principalmente em cães com mais de 5 anos de idade, com uma idade média de 9 anos. Os objetivos do presente estudo foram: avaliar os casos clínicos com suspeita de neoplasia, compilar os dados clínicos e exames complementares e relacioná-los com os achados anátomo-patológicos. Os tumores foram descritos quanto ao aspecto macro e microscópicos, elaborando-se o diagnóstico do neoplasma. Foram empregadas técnicas de imunoístoquímica para complementação das descrições de tais neoplasmas visando estabelecer comparação entre os aspectos morfológicos encontrados na espécie humana. Utilizou-se 14 cães com diagnósticos de tumores intracranianos, que foram avaliados através do exame físico geral e neurológico associado a exames de imagem (tomografia computadorizada, ressonância magnética, ecoencefalografia) e/ou eletrodiagóstico através de um eletroencéfalografo digital com mapeamento cerebral. A sintomatologia depende da localização do tumor, agressividade tumoral, tipo de formação e severidade da lesão associada. Subdividiram-se os cães em 4 grupos de acordo com a sintomatologia: grupo I (cães com alterações cerebrais); grupo II (cães com alterações cerebelares); grupo III (cães com alterações em tronco encefálico); grupo IV (cães com alteração mista). Os achados mais freqüentes nos animais acometidos foram: convulsões, alteração de comportamento, andar compulsivo, andar em círculos, progressão obstinada, déficits proprioceptivos e/ou motores e déficits vestibulares. A confirmação diagnóstica, caracterização histopatológica e classificação das neoplasias foram realizadas através de biopsia ou necropsia dos animais eutanasiados ou que vieram a óbito espontaneamente. / Neurological disorders, namely intracranial tumors, have a great importance among morbid entities in dogs, manly in animals older then five years, with a mean of age ranging nine years old. The aim of this study was to evaluate clinical cases suspected of having neoplasia and to associate ancillary clinical exams with necropsy findings. Tumors were described macro and microscopically, and a histopathological diagnosis was determinated. Immunohistochemical methods were applied to ensure the diagnosis and to compare with morphological data available in human counterparts. A number of 14 dogs with intracranial tumor were used, from this cases compilation of general physical examination, neurological evaluation, and associated image diagnosis (computerized tomography, magnetic resonance and echoencephalography) and/or eletrodiagnois by means of a digital electroencephalographic mapping of brain. The symptoms depend on tumor localization, tumor behavior - aggressivety, type of tumor and severity of associated lesions. Four groups were subdivided according to the symptoms: group I (dogs with brain alterations); group II (dogs with cerebellar alterations); group III (dogs with brain steam alterations) and group IV (dogs with mixt alterations). The most frequent findings in diseased animals were seizures, behavior alterations, compulsive walking, circling, obstinate progression, propioceptive and/or motor deficit and vestibular deficit. The diagnosis confirmation, histopathological characterization and classification of neoplasias were made by biopsy or necropsy of euthanatized or naturally dead animals.

A vareniclina é uma substância química sintética utilizada para o tratamento de tabagismo; atua como agonista de receptores colinérgicos nicotínicos, em especial, como agonista parcial em receptores α4β2 e α3β4, e como agonista total do receptor α7. Levando em consideração que há uma tendência de ampliação do uso clínico da vareniclina para o tratamento da dependência à diversas substâncias de padrão abusivo e que há poucos estudos relacionados aos seus efeitos sobre o comportamento, cognição e sistema motor, tornam-se necessários mais estudos sobre essa substância. Assim, no presente trabalho foram estudados os efeitos da exposição prolongada (28 30 dias) de ratos à vareniclina, avaliando-se o consumo de água e de ração, o ganho de peso e o comportamento animal, por meio dos testes de campo aberto, labirinto em cruz elevado, interação social, comportamento estereotipado, labirinto de Barnes e esquiva passiva. Ainda foram feitas as avaliações dos níveis de neurotransmissores e seus metabólitos em diferentes estruturas cerebrais, bem como avaliações hematológicas, bioquímicas séricas, urinárias e estudos anatomopatológicos e histopatológicos. Foram utilizadas três doses de vareniclina: 0,03 (dose terapêutica para o ser humano), 0,1 e 0,3 mg/kg, por via oral (gavagem). Os resultados mostraram que a exposição prolongada de ratos à diferentes doses de vareniclina não provocou toxicidade, uma vez que não houve alteração no consumo médio de água e de ração e no ganho de peso avaliados semanalmente. Quanto às avaliações comportamentais, observou-se leve aumento da atividade geral no campo aberto, bem como diminuição do tempo de interação social, não sendo capaz de alterar parâmetros neuroquímicos, hematológicos, bioquímicos séricos, urinários, anatomopatológicos e histopatológicos de ratos expostos à vareniclina. / Varenicline is a synthetic chemical used for the smoking addiction treatment; it acts as an agonist of nicotinic cholinergic receptors, in particular, as a partial agonist of receptors α4β2 and α3β4 and as a full agonist of the α7 receptor. More studies about this substance are necessary, given that its clinical use is increasingly being applied to the treatment of addiction to a variety of abusive drugs. Moreover, there are few studies on vareniciline effects on behavior, cognition and the motor system. Thus, in this study the effects of prolonged (28-30 days) exposure of rats to varenicline were evaluated. It was analyzed the water and food consumption, the weight gain and the animal behavior, through open field, elevated plus maze, social interaction, stereotyped behavior, Barnes maze and passive avoidance tests. The neurotransmitter levels and their metabolites in different brain structures were measured and hematological, serum biochemistry, urinary evaluations and pathological and histological studies were carried out. We used three doses of varenicline: 0.03 (therapeutic dose for humans), 0.1 and 0.3 mg/kg orally (gavage). The results showed that prolonged exposure of rats to different doses of varenicline did not cause toxicity, since there were no changes in average weekly consumption of water or food nor body weight gain, which were measured weekly. As for behavioral assessments, there was a slight increase in overall activity in the open field as well as decreased time of social interaction. Varenicline was not able to change neurochemical, hematological, serum biochemical, urinary, pathology and histopathology parameters of rats.

O Tabagismo é uma das principais causas de doenças crônicas não transmissíveis, afetando cerca de 1,6 bilhões de pessoas até 2030. Políticas de controle do tabagismo foram criadas para evitar a contaminação de indivíduos não fumantes pela poluição tabagística ambiental. Descritas principalmente no pulmão, pouco se sabe sobre a ação da fumaça do cigarro no sistema nervoso central (SNC). O presente estudo tem como objetivo avaliar os efeitos da exposição à fumaça do cigarro, por 15 dias consecutivos, em um processo de inflamação sistêmica induzida por LPS. Para tanto, camundongos C57BL/6 foram expostos a uma mistura de fumaça central e lateral do cigarro referência 3R4F (Universidade de Kentucky, EUA). No último dia, os animais foram desafiados com LPS iv. (0,1 µg/animal) ou salina, formando os grupos CO – controle, FU – fumaça do cigarro, LPS – desafio com LPS e FPS – fumaça do cigarro e desafio com LPS. As amostras foram processadas de maneira a realizar as análises de RT-PCR para os primers IL-6, IL-1β, TNF-α, TLR2, TLR4 e iNOS, além da dosagem por ELISA das citocinas IL-6, IL-10, IL-1β e TNF-α e western blot para a proteína P65. Nossos resultados demonstraram um aumento da transcrição dos genes TLR2, TLR4 e iNOS, em todas as estruturas analisadas no período de 2 horas de eutanásia após o desafio com LPS no grupo FPS em relação a todos os outros grupos estudados. Já no período de 4 horas após o desafio foi possível observar um aumento dos genes IL-1β e TNFα em ambos grupos que receberam o desafio com o LPS no hipocampo, estriado, córtex e cerebelo, sugerindo que a influência da fumaça do cigarro se de apenas 2 horas após o desafio, uma vez que no período de 4 horas observamos apenas o efeito do LPS. Os resultados da dosagem de citocinas sugerem que, no período de 4 e 6 horas após o desafio, a citocina anti-inflamatória IL-10 está diminuída no hipocampo e no córtex pré-frontal no grupo FPS em relação a todos os outros grupos estudados. Porém é possível observar um aumento estatisticamente significativo da mesma citocina no cerebelo para o grupo exposto apenas à fumaça do cigarro. Nossos dados sugerem que o processo inflamatório no SNC ocorra de maneira diferenciada dependendo da região estudada, e do período analisado. / Cigarette smoking is a major cause of chronic non-communicable diseases, affecting approximately 1.6 billion people up to 2030. Tobacco control policies were created in order to prevent contamination of non-smokers by environmental tobacco smoke. Described primarily in the lung, little is known about the action of cigarette smoke in the central nervous system (CNS). The present study aims to evaluate the effects of cigarette smoke exposure for 15 consecutive days in a process of systemic inflammation induced by LPS. To this so, C57BL/6 mice were exposed to a mixture of mainstream and sidestream cigarette smoke reference 3R4F (University of Kentucky, USA). In the last day, animals were challenged with LPS iv. (0.1 µg/animal) or saline and divided into the following groups: CO - control, FU - cigarette smoke, LPS - LPS challenge and FPS - cigarette smoke and LPS challenge. The samples were processed in order to perform the RT-PCR analysis for IL-6, IL-1β, TNF-α, TLR2, TLR4 and iNOS primers, ELISA assay of IL-6, IL-10, IL-1β and TNF-α and western blot for p65. Our results showed an increase in transcription of TLR2, TLR4 and iNOS genes in all structures analyzed within 2 hours of euthanasia after LPS challenge in the FPS group compared to all other groups. Beside this 4 hours after challenge was observed an increase of IL-1β and TNF genes in both groups that received the challenge with LPS in the hippocampus, striatum, cortex and cerebellum, suggesting that the influence of cigarette smoke can be observed only 2 hours after the challenge, justified since in a time period of 4 hours we can only observe the effect of LPS. The results suggest that cytokine assay, between 4 and 6 hours after challenge, the anti-inflammatory cytokine IL-10 is decreased in FPS group in the hippocampus and prefrontal cortex. But it is possible to observe a statistically significant increase in the same cytokine in the cerebellum exposed only to cigarette smoke group. Our data suggest that the inflammatory process in the CNS occurs differently depending on the region studied, and the period analyzed.

As enfermidades neurológicas, notadamente os tumores intracranianos, têm grande importância dentre os quadros mórbidos que acometem animais da espécie canina, principalmente em cães com mais de 5 anos de idade, com uma idade média de 9 anos. Os objetivos do presente estudo foram: avaliar os casos clínicos com suspeita de neoplasia, compilar os dados clínicos e exames complementares e relacioná-los com os achados anátomo-patológicos. Os tumores foram descritos quanto ao aspecto macro e microscópicos, elaborando-se o diagnóstico do neoplasma. Foram empregadas técnicas de imunoístoquímica para complementação das descrições de tais neoplasmas visando estabelecer comparação entre os aspectos morfológicos encontrados na espécie humana. Utilizou-se 14 cães com diagnósticos de tumores intracranianos, que foram avaliados através do exame físico geral e neurológico associado a exames de imagem (tomografia computadorizada, ressonância magnética, ecoencefalografia) e/ou eletrodiagóstico através de um eletroencéfalografo digital com mapeamento cerebral. A sintomatologia depende da localização do tumor, agressividade tumoral, tipo de formação e severidade da lesão associada. Subdividiram-se os cães em 4 grupos de acordo com a sintomatologia: grupo I (cães com alterações cerebrais); grupo II (cães com alterações cerebelares); grupo III (cães com alterações em tronco encefálico); grupo IV (cães com alteração mista). Os achados mais freqüentes nos animais acometidos foram: convulsões, alteração de comportamento, andar compulsivo, andar em círculos, progressão obstinada, déficits proprioceptivos e/ou motores e déficits vestibulares. A confirmação diagnóstica, caracterização histopatológica e classificação das neoplasias foram realizadas através de biopsia ou necropsia dos animais eutanasiados ou que vieram a óbito espontaneamente. / Neurological disorders, namely intracranial tumors, have a great importance among morbid entities in dogs, manly in animals older then five years, with a mean of age ranging nine years old. The aim of this study was to evaluate clinical cases suspected of having neoplasia and to associate ancillary clinical exams with necropsy findings. Tumors were described macro and microscopically, and a histopathological diagnosis was determinated. Immunohistochemical methods were applied to ensure the diagnosis and to compare with morphological data available in human counterparts. A number of 14 dogs with intracranial tumor were used, from this cases compilation of general physical examination, neurological evaluation, and associated image diagnosis (computerized tomography, magnetic resonance and echoencephalography) and/or eletrodiagnois by means of a digital electroencephalographic mapping of brain. The symptoms depend on tumor localization, tumor behavior - aggressivety, type of tumor and severity of associated lesions. Four groups were subdivided according to the symptoms: group I (dogs with brain alterations); group II (dogs with cerebellar alterations); group III (dogs with brain steam alterations) and group IV (dogs with mixt alterations). The most frequent findings in diseased animals were seizures, behavior alterations, compulsive walking, circling, obstinate progression, propioceptive and/or motor deficit and vestibular deficit. The diagnosis confirmation, histopathological characterization and classification of neoplasias were made by biopsy or necropsy of euthanatized or naturally dead animals.

Functional magnetic resonance imaging (fMRI) can be used to track neural activation in the brain during functional activities. The purpose of this study was to investigate brain neural responses to blood flow restricted (BFR) versus control handgrip exercise. Using a randomized crossover design, 25 subjects (12 males, 13 females) completed handgrip exercises during two conditions: BFR vs. control. To familiarize participants with the exercise conditions, one week prior to MRI scanning participants completed each exercise condition once on separate days, with 72 hours between days. The following week fMRI scans were performed at the same time of day, separated by 72 hours. The exercise protocol consisted of five 30-second sets of squeezing a nonmetallic handgrip exerciser (a reported 13.6 kg resistance), doing as many repetitions as possible, with 20-second rest intervals between sets. We saw a significant main effect of exercise condition (BFR versus control) between premotor dorsal (PMd)(F = 5.71, p = 0.022), premotor ventral (PMv)(F = 8.21, p = 0.007), and right ventral striatum (VS_R)(F = 7.36, p = 0.01). When considering anatomical regions of interest, we did not find significant differences between exercise conditions in bilateral S1 (p > 0.82), primary motor cortex (M1)(p > 0.33), supplementary motor area (SMA)(p > 0.66), cerebellum (CB)(p > 0.70), insular cortex (INS)(p > 0.45), anterior cingulate cortex (ACC)(p > 0.24), or thalamus (TH)(p > 0.66). Bilateral ACC (ACC_B), right middle frontal gyrus (MFG_R), and the right primary sensory cortex (S1_R) showed significant linear trends (p = 0.001) over the five exercise sets. Finally, the S1_R, left primary sensory cortex (S1_L), and the right anterior cingulate cortex (ACC_R) showed a main effect of set (p < 0.02). These data demonstrate that acute training with BFR during handgrip exercise results in different neural activation patterns in select areas of the brain, compared to a control. These results show that while completing less work with BFR exercise, subjects can achieve a similar amount of brain neural activation as with a higher-volume exercise. Brain neural activation is important to overall patient health and these findings may be important for prescribing training with BFR in clinical and applied research settings.