Teses em Neurociências e Biologia Celular (Doutorado) - PPGNBC/ICB

URI Permanente para esta coleçãohttps://repositorio.ufpa.br/handle/2011/2390

O Doutorado Acadêmico pertence ao Programa de Pós-Graduação em Neurociências e Biologia Celular (PPGNBC) do Instituto de Ciências Biológicas (ICB) da Universidade Federal do Pará (UFPA).

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Acesso aberto (Open Access)
Acidente vascular encefálico isquêmico na exposição crônica ao etanol: estudo pré-clínico da comorbidade e da resposta a minociclina
(Universidade Federal do Pará, 2015-02-27) FONTES JÚNIOR, Enéas de Andrade; MAIA, Cristiane do Socorro Ferraz; http://lattes.cnpq.br/4835820645258101; CRESPO LÓPEZ, Maria Elena; http://lattes.cnpq.br/9900144256348265
Stroke is the second largest cause of death in the world and the leading in Brazil, with 87% of strokes due to ischemic processes. Chronic ethanol consumption, usually beginning in adolescence, is recognized as an independent risk factor for increased morbidity and mortality by stroke. Although cases combining the two diseases are relatively common, there is no data in animals or clinical models demonstrating the quality or mechanisms of interaction between the two morbidities, nor its impact on therapeutic intervention. Considering the recent studies proposing minocycline as a new therapeutic tool for the treatment of stroke, this study aimed to investigate the interaction between the Chronic Alcoholic Intoxication (CAI) started in adolescence and the stroke in motor cortex of adult rats, and the effects of treatment with minocycline on this interaction, using behavioral, cellular and molecular parameters. Female Wistar rats (35 days-old) were chronically exposed to ethanol (6.5 g/kg/day, 22.5% w/v) or water for 55 days. One day after the end of the CAI focal ischemia was induced in motor cortex with the endothelin-1 (ET-1), followed by seven-day treatment with minocycline or saline. After this period, the animals were assayed with open field and rota rod tests. Immediately, animals were sacrificed and cortex was dissected for evaluation of nitrite and lipid peroxidation levels. In all groups, some animals were perfused and the motor cortex subjected to histological analysis to assess the damage, and immunohistochemical labeling to neuronal death (anti-NeuN), microglial/macrophage (anti-ED1) and astrocytes (anti-GFAP) activation. The ethanol intoxication from puberty to adulthood potentiated the damage caused by stroke, causing major losses in capacity to start and running movements as well as the strength and motor coordination compared to ischemic animals pretreated with water. These manifestations were accompanied by increased neuronal loss, reduced ED-1+ and GFAP+ cells and higher levels of nitrite and lipid peroxidation. Treatment with minocycline was effective in preventing/reverse motor deficits and tissue damage induced by focal ischemia, also inhibiting the increase in oxidative stress markers. The CAI either alone with succeeded by focal ischemia, harmed the outcome of treatment with minocycline. Our results indicate that heavy alcohol intoxication during adolescence exacerbates the motor deficit and tissue damage in animals subjected to focal ischemia. This process appears to be associated with microglia/astroglial activation, but mainly with oxidative stress. It also shows that the previous history of CAI started adolescence interferes significantly in the treatment of cerebral ischemia with minocycline.
Acesso aberto (Open Access)
Análise comparativa dos padrões neurodegenerativos da substância cinzenta em diferentes áreas corticais de ratos adultos submetidos à lesão isquêmica focal
(Universidade Federal do Pará, 2012-09-27) SANTOS, Enio Maurício Nery dos; LEAL, Walace Gomes; http://lattes.cnpq.br/2085871005197072
Stroke can occur in any region of the central nervous system (CNS). The cerebral cortex is one of the most often affected areaby this acute neural disorder, but there are no studies that have compared the damaging pattern in different cortical regions after acomparable focal ischemia. The aim of this investigation was to evaluate the degenerative pattern of different cortical areas after focal ischemic injury. Focal ischemia was induced by stereotaxic microinjections of endothelin-1 (ET-1) into the somatosensory, motor and association cortices of adult rats (N = 45). The control animals were injected with the same volume of sterile saline (N = 27). The animals were perfused 1, 3 and 7 days after the ischemic event. The brain was removed, postfixed, cryoprotected, and sectioned in a cryostat. The general histopathology was evaluated in 50μm sections stained with cresyl violet. 20μm sections were submitted to immunohistochemistry for astrocytes (anti-GFAP), activated microglia / macrophages (anti-ED1) and overall microglial population (anti-Iba1). The damaging patterns werequalitatively evaluated under optical microscopy and quantitatively by counting the number of cells in the ipsilateral and contralateral sides to injury.Descriptive statistics and comparisons within and between groups were performed using analysis of variance with Tukey post-hoc test. Conspicuous ischemic tissue loss, microglial activation and astrocytosis were observed mainly 3 and 7 days after ischemia, which was not observed in control animals. The tissue loss and activation of glial cells were more intense in the somatosensory cortex, followed by the motor cortex. The association cortex displayed less damage compared to other cortical areas, which was confirmed by quantitative analysis. The results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas.
Acesso aberto (Open Access)
Ativação microglial, lesão da substância branca e expressão de Nogo-A em ratos submetidos à isquemia estriatal
(Universidade Federal do Pará, 2012-05-10) LIMA, Rafael Rodrigues; LEAL, Walace Gomes; http://lattes.cnpq.br/2085871005197072
The objective of this investigation was to evaluate the degenerative pattern of several white matter tracts after striatal ischemic injury, correlating degenerative process standards with the microglial activation and expression of Nogo-A. For this purpose, focal ischemia was induced with stereotactic injection of endothelin in striatum of adult rats, and only in the control animals injected with sterile saline. The animals were perfused 3, 7, 14 and 30 days after ischemia. The brain removed, postfixed, cryoprotected, cut into cryostat sections obtained and submitted to immunohistochemical investigation with the following antibodies: anti-GFAP (1:2000, Dako), anti-Tau-1 (1:500, Chemicon), Anti-MBP (1:100, Chemicon International), Anti-Nogo-A (1:100, Invitrogen), Anti-Iba1 (1:1000, WAKO), ED1 (1:500, Serotec) and Anti-MHC II (Abcam 1:100), besides the viewing of the damage pattern with cresyl violet. Slides are marked by different methods were evaluated qualitatively and quantitatively also some (Anti-Nogo A, anti-ED-1, anti-MHC-II and anti-tau-1), counts were carried out in the striatum and in the corpus callosum. The data were tabulated, statistically analyzed by Tukey test (p <0.05) and micrographs taken of the findings more representative. The slides were stained with cresyl violet revealed an increase in cell density by the infiltration of inflammatory cells to the ischemic area, with a significant increase on day 7. The blades immunostained for GFAP was found progressive increase of the population of astrocytes and an increase in cell volume 7 and 14 days. Oligodendrocyte pathology marked with Tau-1 had peak marking the 3rd day in the striatum and the 7th day in the corpus callosum, and loss of myelin compaction identified by MBP was better at 14, in the different treatment. The microglial activation identified by different immunoblots showed a peak on day 7, both in striatum and in the corpus callosum, but in the corpus callosum with a much smaller number compared to the striatum. The morphology of microglial underwent changes, which found the branched phenotype in control animals, as well as in early and late times after ischemia and amoeboid default / phagocytic day 7, coinciding with the largest number of activated cells. The count of Nogo-A + cells peaked at 3 days observed in the striatum, and there were no differences in the corpus callosum expression Nogo-A 3 to 14 days, only a decrease compared to 30 days. Thus, microinjections of ET-1 induced conspicuous striatal tissue loss, concomitant with progressive microglial activation, astrocytosis, loss of immunoreactivity for myelin basic protein and oligodendrocytes damage in various survival times after focal ischemia. These events affect a few SB tracts, as the corpus callosum. The establishment of the temporal evolution of these events is the neuropathological basis for future studies, in which they should handle the inflammatory response in order to minimize these tissue changes.
Acesso aberto (Open Access)
Efeitos anti-inflamatórios e neuroprotetores do extrato de gergelim preto (Sesamum indicum L.) em um modelo experimental de isquemia estrial
(Universidade Federal do Pará, 2024-03) SANTOS, Ijair Rogério Costa dos; LEAL, Walace Gomes; http://lattes.cnpq.br/2085871005197072
Acidente Vascular Encefálico (AVE) é uma neuropatologia caracterizada como o surgimento súbito global ou focal de déficits da função neurológica de duração superior a 24 horas ou que leve a morte, cuja única causa reside na origem vascular. Estudos sobre a incidência, comprometimento físico e mortalidade enquadram o AVE como a segunda causa de morte no mundo e a principal complicação orgânica que leva às disfunções físico-neurológicas, frequentemente, graves e permanentes. A indução do AVE em animais de experimentação e o entendimento de sua fisiopatologia, bem como a busca de tratamentos que minimizem os danos neurológicos e estimulem a recuperação morfofuncional do indivíduo afetado são temas de grande relevância científica e clínica. Neste estudo, investigamos os possíveis efeitos neuroprotetores e/ou anti-inflamatórios do extrato supercrítico de gergelim preto (Sesamun indicum L.) após lesão isquêmica focal por microinjeções de 80 pmol de endotelina-1 no estriado de ratos adultos, usando as coordenadasestereotáxicas: 1,2 mm, anterior-posterior; 2,5 mm, médio-lateral; 4,0 mm, dorsoventral. Após a indução do AVE, os grupos controles foram tratados com tween a 5% e os tratados receberam 150 mg/kg de gergelim, ambos, por via intraperitoneal, em duas doses diárias de 75 mg/kg. A neuropatologia foi obtida em secções encefálicas com 50 e 20 μm de espessuras e coradas com violeta de cresila, para identificar a área de lesão, e/ou imunomarcadas por anticorpos específicos à identificação de neurônios (anti-NeuN), astrócitos (anti-GFAP) e micróglia (anti-ED1). Secções de 5 μm de espessura de rim e fígado corados por métodos histológicos e histoquímicos não mostraram alterações morfológicas nas células que compõem esses órgãos essenciais, sugerindo baixa toxicidade do extrato. Todas as secções coradas e/ou imunomarcadas foram visualizadas em microscópio óptico e seuscampos mais ilustrativos, em todos os tempos de sobrevida e grupos experimentais,foram capturados digitalmente e editados em computador. A quantificação das célulasNeuN+(neurônios), micróglia/macrófagos (ED1+) e astrócitos (GFAP+) na área de lesão, três secções por lâmina, todo campo ao redor de lesão por secção, com auxílio de uma gradícula de área 0,0625 mm2 na ocular possibilitou o teste t-Student à análise estatística entre os grupos e o uso do programa Microsoft Excel à plotagem dos gráficos. Por fim, uma caracterização da citotoxicidade in vitro, bem como a verificação do índice de acidez do extrato revelou baixa acidez e mínima agressividade em células sanguíneas, que ratifica o uso do extrato supercrítico em estudos que visem otratamento de doenças agudas e crônicas no SNC.
Acesso aberto (Open Access)
Imunoreatividade para os receptores de neurotrofinas P75NTR e TrkA na zona subventricular de ratos adultos após isquemia estriatal
(Universidade Federal do Pará, 2015-08-21) TAVARES, Patrycy Assis Noronha; LIMA, Rafael Rodrigues; http://lattes.cnpq.br/3512648574555468; LEAL, Walace Gomes; http://lattes.cnpq.br/2085871005197072
Neurotrophins are growth factors expressed by cells of the nervous system both during development and in adulthood. The Nerve Growth Factor (NGF, the English- Nerve Growth Factor), brain-derived neurotrophic factor (English- BDNF- of Brain-Derived Neurotrophic Factor), Neurotrophin-3 (NT-3), Neurotrophin-4/5 ( NT-4/5), have many functions related to aging and response of nervous tissue to the pathology such as vascular accident (CVA). In this pathology, the increase of the neurotrophin expression can interfere with the degree of neurogenesis in the sub-ventricular zone (SVZ) and redirect the rostral migratory flow of Adult Neural Stem Cells (CTNAs) to the ischemic region. The presence of neurotrophin receptors TrkA and p75NTR in the CTNAs of SVZ indicates that they may participate in the regulation of neurogenesis in this region. Here we describe the influence of an experimental ischemia by microinjection of a vasconstritor Endothelin-1 peptide, which is restricted to the striatum adjacent SVZ; on the pattern of immunoreactivity for TrkA and p75NTR receptors in different survival times. The histopathological pattern of ischemic striatum and the cytoarchitecture of the SVZ, followed by immunohistochemical analysis to the receptors were analyzed. Numerous p75NTR + cells were found in the ipsilateral SVZ and against the injection site, with had a reduction in immunoreactivity at first and third day after ischemia. Few TrkA + cells were found in SVZ of both groups, however, many TrkA + axonal terminals were saw in the ischemic ipsilateral SVZ. Soon after the ischemic process, there was thickening of the SVZ, the concomitant reduction in immunoreactivity for p75NTR and TrkA + arisings of axonal terminals.
Acesso aberto (Open Access)
Modulação da neuroinflamação celular e neuroproteção induzidas por tratamento com betacariofileno em um modelo experimental de isquemia estriatal em ratos adultos
(Universidade Federal do Pará, 2016-10-11) LOPES, Rosana Telma Santos; SANTOS, Enio Maurício Nery dos; http://lattes.cnpq.br/7789458294239924; LEAL, Walace Gomes; http://lattes.cnpq.br/2085871005197072
Stroke results from the transitory or permanent reduction of cerebral blood flow. It can be classified as hemorrhagic or ischemic. Ischemic stroke is responsible for around 87% of all cases. This acute neural disorder is the second cause of mortality and disability around the world and the main cause of death in Brazil. Since ischemic stroke in patients usually results from a thrombotic or embolic occlusion of the middle cerebral artery (MCA), experimental models of ischemia have been developed to mimic human stroke. There are no neuroprotective drugs available for human stroke. It follows that research on development of alternative neuroprotective drugs are of important clinical relevance. In this study, we investigated the effects of betacaryophyllene, the main sesquiterpene present in about 40% of the copaiba oil-resin composition, on microglial activation, astrocytic reactivity and neuronal preservation following experimental MCAO in adult rats. Animals were submitted to experimental stroke by microinjections of endothelin-1 (ET-1) and treated (i.p) with betacaryophillene (N=4) or vehicle control (N=4) and perfused at 3 days or 7 days post-MCAO. Gross histopathology was performed using cresyl violet staining. Immunohistochemistry was used to assess neuronal loss (anti-NeuN), microglial activation (anti-ED1) and astrocytosis (anti-GFAP). Numbers of NeuN+ and GFAP+ cells were quantified in the ischemic striatum. Betacaryophyllene treatment reduced microglial activation, increased neuronal preservation and decreased astrocytic reactivity at 7 days post-MCAO. These results suggest that betacaryophylene modulates neuroinflammation and is neuroprotective following experimental striatal. Considering that betacaryophyllene is a natural dietetic extract already used in non-neural human diseases with antiinflammatory, anti-microbial and anti-carcinogenic properties, its use as a neuroprotective agent is a promising future therapy for human stroke.

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