Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 128-139). Also available online.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Centre of Neuroscience. Title from pdf file main screen (viewed on April 18, 2010). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 161 pages. Includes Vita. Includes bibliographical references.

Remyelination describes the regeneration of myelin sheaths, and is considered one of the most promising strategies for improving the prognosis of demyelinating diseases such as multiple sclerosis. Data from animal models and human studies have shown that remyelination can occur extensively in the central nervous system (CNS), leading to functional recovery and axonal protection. However, remyelination does not always proceed to completion, and its failure is associated with progressive neurological disability. Thus, there is clinical need for interventions that can optimise the conditions for remyelination. Recent advances in genomics and animal husbandry have kindled an interest in the microbiome as a means to influence processes throughout the body. Our commensal microbes communicate with host cells at epithelial barriers, stimulate neural and endocrine axes and directly produce a plethora of long-range signalling molecules. Critically, the development and maintenance of the immune system depend on signals from the microbiota, and we know that a well-coordinated immune response is a key determinant of the success of remyelination. This thesis explores how the microbiome can influence CNS remyelination. To do so, I have studied remyelination in three murine models of microbiome alteration. Firstly, long-term oral administration of an antibiotic cocktail was used to deplete the microbiota of adult mice. Following focal demyelination, these mice had deficits in their inflammatory response, clearance of myelin debris and differentiation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Faecal microbial transplant was able to rescue aspects of the inflammatory response and phagocytosis, but not OPC differentiation. Secondly, I looked at remyelination in germ-free (GF) mice following cuprizone-induced demyelina- tion. As with the antibiotics-treated mice, there were deficits in inflammation following demyelination, which tended to peak later than in control mice. Finally, I investigated the potential of a therapeutic probiotic (VSL#3) to improve remyelination in aged mice. In contrast to antibiotic treatment, probiotic administration caused a slight enhancement in the onset of inflammation following focal demyelination. However, there was no significant improvement in OPC differentiation or toluidine blue rank analysis, suggesting these changes in inflammation were not sufficient to positively modulate remyelination. The results from these three studies introduce a significant but previously unconsidered environmental influence on remyelination in the CNS. Whilst the effects are subtle relative to more direct interventions, the microbiome can be manipulated simply and non-invasively, which may provide a useful adjunct to other strategies for optimising remyelination.

A hemorragia intracerebral (HIC) é uma das principais causas de mortalidade em todo mundo, desencadeando eventos adversos e déficits neurológicos graves. A partir disso, essa tese teve como objetivo avaliar as respostas celulares e suas interações envolvidas na HIC e investigar os possíveis mecanismos que contribuem para os déficits sensório-motores relacionados, principalmente, à habilidade dos membros anteriores dos animais. Para isso foram realizados dois experimentos. Em ambos os animais de 90 dias foram submetidos à HIC induzida por colagenase no estriado dorsolateral. No primeiro experimento, após 24h, 72h e 7 dias os animais foram avaliados funcionalmente, e histologicamente quanto ao volume da lesão. Ainda, durante 6h, 24h, 72h e 7 dias, o estriado e o córtex foram avaliados quanto ao número de células neuronais, gliais, e em apoptose; a expressão de GFAP e S100B, no estriado e no córtex, a secreção de S100B, no soro e líquido cérebro-espinhal (LCE). A HIC provocou déficits sensório-motores e perda tecidual; induziu morte neuronal e gliose reativa no estriado. O córtex obteve uma resposta astrocitária similar, mas quanto à microglia houve um aumento apenas em 6h. A S100B correlacionou-se positivamente com o número de células gliais; e negativamente com a força e função dos membros anteriores dos animais lesados. No segundo experimento, foram avaliados o estado oxidativo, o nível de TNF-α, a atividade da glutamina sintetase (GS) e a captação de glutamato nos tempos de 6h, 24h, 72h e 7 dias pós-lesão. Após 6h da HIC houve aumento no nível de TNF-α e excitotoxicidade glutamatérgica; entretanto, a função de clearence astrocitário foi restabelecida 7 dias após o dano. A HIC gerou estresse oxidativo e aumento de antioxidantes endógenos, além disso, as espécies reativas de oxigênio (ERO) inibiram a atividade da GS em 24h e 7 dias após a lesão. A partir dos resultados obtidos, podemos concluir que a S100B atua na lesão hemorrágica produzindo respostas distintas, região dependente, contribuindo para o aumento das células gliais no estriado e córtex, correlacionando-se positivamente com os déficits funcionais. Este é o primeiro relato descrevendo a relação da S100B com as alterações celulares e funcionais durante a progressão da HIC experimental. Além disso, os mecanismos envolvidos na HIC demonstraram padrões e respostas envolvendo excitotoxicidade glutamatérgica e inflamação, que colaboraram para o dano oxidativo; em resposta ao estresse houve o aumento de antioxidantes endógenos que podem ter contribuído para o reestabelecimento da função astrocitária. / Intracerebral haemorrhage (ICH) is a major cause of mortality worldwide, triggering adverse events and serious neurological deficits. From this standpoint, this thesis aimed to evaluate the cellular responses and interactions involved in ICH and investigate the possible mechanisms that contribute to sensory-motor deficits, mainly related to the ability of the forelimbs of animals. For this, two experiments were conducted. In both, animals of 90 days of age were subjected to ICH induced by collagenase in dorsolateral striatum. In the first experiment, after 24h, 72h and 7 days, the animals were evaluated functionally, and histologically about the lesion volume. Also, at 6h, 24h, 72h and 7 days, the striatum and the cortex was evaluated as the number of neuronal, glial and apoptotic cells; the expression of GFAP and S100B, the striatum and cortex, S100B secretion in serum and cerebrospinal fluid (CSF). ICH caused sensorimotor deficits and tissue loss; induced neuronal death and reactive gliosis in the striatum. The cortex astrocyte obtained a similar response, but about the microglia there was an increase only at 6h. S100B was positively correlated with the number of glial cells; and negatively correlated with the strength and function of the forelimbs of injured animals. In the second experiment, were evaluated the oxidative state, the level of TNF-α, the activity of glutamine synthetase (GS) and glutamate uptake at 6h, 24h, 72h and 7 days post-injury. After 6h from ICH, there was an increase in TNF-α levels and glutamatergic excitotoxicity; however, the astrocyte clearance function was established 7 days after the damage. HIC generated oxidative stress and increase of endogenous antioxidants, in addition, reactive oxygen species (ROS) inhibited the activity of GS in 24h and 7 days after injury. Based on the results obtained, we can conclude that S100B acts in hemorrhagic lesions producing different responses, region-dependent, contributing to the increase in glial cells in the striatum and cortex, correlating positively with functional deficits. This is the first report describing the relation of S100B with cellular and functional changes during the progression of experimental ICH. Furthermore, the mechanisms involved in ICH displayed patterns and responses involving glutamatergic excitotoxicity and inflammation, that contributed to oxidative damage; in response to stress there was an increase of endogenous antioxidants which may have contributed to the re-establishment of astrocyte function.

Compostos antioxidantes e anti-inflamatórios vêm sendo apontados como uma alternativa para auxiliar no tratamento da epilepsia. Ácido rosmarínico (AR) e seu metabolito ácido caféico (AC) têm ação antioxidante e anti-inflamatória relatada em vários estudos. Além disso, AR tem ação inibitória sobre GABA transaminase (GABA-T), enzima que metaboliza um dos principais neurotransmissores envolvidos na epilepsia: o GABA. Nesta tese, investigamos o efeito do AR e AC em modelos de convulsão e sua ação neuroprotetora sobre o estresse oxidativo, neuroinflamação e genotoxicidade relacionados à epilepsia. Para isso, utilizou-se primeiramente o modelo de kindling induzido por pentilenotetrazol (PTZ) para avaliar o efeito dos compostos na epileptogênese. Os compostos não produziram efeito antiepileptogênico in vivo neste modelo, no entanto, algumas doses de AR e AC mostraram um efeito neuroprotetor contra o estresse oxidativo e contra o dano ao DNA induzidos pelo kindling no córtex dos camundongos, demostrando um efeito benéfico contra alterações fisiológicas associadas à epileptogênese. Baseados no fato do AR ser um inibidor da GABA-T, avaliamos o efeito dos compostos na potencialização da ação do agonista GABAérgico diazepam (DZP) sobre as convulsões agudas induzidas por PTZ e pilocarpina (PIL) e no teste do sono induzido por DZP. O efeito de AR e AC na prevenção do dano genotóxico produzido pelos modelos também foi investigado. AR e AC mostraram potencializar a ativação GABAérgica, o que foi evidenciado pelo aumento da latência para a convulsão aguda induzida por PTZ, promovido por AR (4 mg/kg), e pela redução para o início do sono induzido por DZP, promovido por AR (4 mg/kg) e AC (8 mg/kg). Além disso, AR e AC (4 mg/kg) reduziram o dano genotóxico causado por PIL. Complementarmente, verificamos (in vitro) que o pré-tratamento com AR foi capaz modular a ativação microglial, produzindo um padrão de ativação anti-inflamatório, reduzindo a formação de espécies reativas e a expressão de citocinas pró-inflamatórias, e o dano genotóxico induzido pela inflamação. Estas respostas foram relacionadas à possível inibição da via apoptótica por AR. Em suma, os trabalhos aqui apresentados evidenciaram um efeito neuroprotetor de AR e AC por reduzir o estresse oxidativo, a ativação de vias inflamatórias e o dano genotóxico, envolvidos na progressão do processo epiléptico. / Antioxidants and anti-inflammatory compounds have been identified as an alternative to improve the treatment of epilepsy. The rosmarinic acid (RA) and its metabolite caffeic acid (CA) have their antioxidant and anti-inflammatory effects reported in several studies. Moreover, AR has inhibitory action on GABA transaminase (GABA-T), the enzyme that metabolizes an important neurotransmitter involved in epilepsy: GABA. In this thesis, we investigated the effect of RA and CA in seizure models and its neuroprotective action on oxidative stress, neuroinflammation and genotoxicity related to epilepsy. For this, first we used the kindling model induced by pentylenetetrazole (PTZ-induced kindling) for evaluating the effect of compounds on epileptogenesis. The compounds had no effect antiepileptogenic in vivo in this model, however, some doses of RA and CA showed a neuroprotective effect against oxidative stress and DNA damage induced by kindling in the cortex of the mice, demonstrating a beneficial effect against physiological changes related to epileptogenesis. Based on the fact of RA being an inhibitor of GABA-T, we evaluated the effect of compounds over potentiation of action of GABAergic agonist diazepam (DZP) on acute seizures induced by PTZ and pilocarpine (PIL) and in DZP-induced sleep test. The preventive effect of genotoxic damage induced by acute seizure models also was investigated. RA and CA showed potentiate GABAergic activation, which was evidenced by increase in latency to acute seizures PTZ-induced promoted by RA (4 mg/kg), and the reduction in latency to sleep in DZP-induced sleep test promoted by RA (4 mg/kg) and AC (8 mg/kg). In addition, RA and CA (4 mg/kg) reduced the genotoxic damage caused by PIL. Moreover, we found (in vitro) that the pre-treatment with RA was able to modulate microglial activation, producing a pattern of anti-inflammatory activation, reducing the formation of reactive species and the expression of proinflammatory cytokines, consequently reducing genotoxic damage induced by inflammation. These responses were related to the possible inhibition of apoptotic pathway by RA. In short, the works presented here showed a neuroprotective effect of RA and CA because of redution of oxidative stress, inflammatory pathways and genotoxic damage, involved in the progression of the epileptic process.

Abnormal activation of brain microglial cells is widely implicated in the pathogenesis of schizophrenia. The disrupted balance of microglia phenotypes has been hypothesized to influence the clinical course of the disease and affect symptom severity. Previously, the pathophysiology of microglial activation was considered to be intrinsic to the central nervous system. We hypothesised that due to their perivascular localization, microglia can also be activated by factors present in circulating blood. We applied a high-content functional screening platform, to characterize alterations in microglial intracellular signalling cascades induced by schizophrenia patient serum relative to control serum. Using automated sample preparation, fluorescent cellular barcoding and flow cytometry, the applied platform is capable of detecting multiple parallel cell signalling responses in microglia. First, we exposed a human microglia cell line to serum isolated from first-onset drug-naïve schizophrenia patients (n=60) and healthy controls (n=79). We were able to show that peripheral blood serum obtained from schizophrenia patients induced differential microglial cell signalling network responses in vitro. We subsequently assessed whether antipsychotic drug-treatment can normalise the abnormal microglial signalling responses previously identified by exposing microglia cells to serum from antipsychotic treated schizophrenia patients (n=15) and controls (n=17). In addition, in order to assess microglia activation in vivo, we obtained positron emission tomography (PET) imaging data from collaborators, who used a radiotracer to assess potential altered microglia activation in patients suffering from schizophrenia. Finally, as a proof of concept study, we attempted to validate these findings by assessing the effect of serum collected from first-onset drug-naïve schizophrenia patients (n=9), controls (n=12) as well as serum isolated from the same patients subjected to six weeks of clinical treatment with the antipsychotic olanzapine (n=9). This study aimed to identify normalisation of previously detected differences in microglia signalling pathways based on successful in vivo treatment. We demonstrate that peripheral blood serum isolated from schizophrenia patients, independent of their treatment status, is sufficient to trigger microglial cell signalling network responses in vitro, which are indicative of altered STAT3 signalling. We further explored the composition of the serum for differential expression of analytes, previously associated with neuropsychiatric disorders, and the utility of the detected microglial cellular phenotype as a target for novel drug discovery.

Compostos antioxidantes e anti-inflamatórios vêm sendo apontados como uma alternativa para auxiliar no tratamento da epilepsia. Ácido rosmarínico (AR) e seu metabolito ácido caféico (AC) têm ação antioxidante e anti-inflamatória relatada em vários estudos. Além disso, AR tem ação inibitória sobre GABA transaminase (GABA-T), enzima que metaboliza um dos principais neurotransmissores envolvidos na epilepsia: o GABA. Nesta tese, investigamos o efeito do AR e AC em modelos de convulsão e sua ação neuroprotetora sobre o estresse oxidativo, neuroinflamação e genotoxicidade relacionados à epilepsia. Para isso, utilizou-se primeiramente o modelo de kindling induzido por pentilenotetrazol (PTZ) para avaliar o efeito dos compostos na epileptogênese. Os compostos não produziram efeito antiepileptogênico in vivo neste modelo, no entanto, algumas doses de AR e AC mostraram um efeito neuroprotetor contra o estresse oxidativo e contra o dano ao DNA induzidos pelo kindling no córtex dos camundongos, demostrando um efeito benéfico contra alterações fisiológicas associadas à epileptogênese. Baseados no fato do AR ser um inibidor da GABA-T, avaliamos o efeito dos compostos na potencialização da ação do agonista GABAérgico diazepam (DZP) sobre as convulsões agudas induzidas por PTZ e pilocarpina (PIL) e no teste do sono induzido por DZP. O efeito de AR e AC na prevenção do dano genotóxico produzido pelos modelos também foi investigado. AR e AC mostraram potencializar a ativação GABAérgica, o que foi evidenciado pelo aumento da latência para a convulsão aguda induzida por PTZ, promovido por AR (4 mg/kg), e pela redução para o início do sono induzido por DZP, promovido por AR (4 mg/kg) e AC (8 mg/kg). Além disso, AR e AC (4 mg/kg) reduziram o dano genotóxico causado por PIL. Complementarmente, verificamos (in vitro) que o pré-tratamento com AR foi capaz modular a ativação microglial, produzindo um padrão de ativação anti-inflamatório, reduzindo a formação de espécies reativas e a expressão de citocinas pró-inflamatórias, e o dano genotóxico induzido pela inflamação. Estas respostas foram relacionadas à possível inibição da via apoptótica por AR. Em suma, os trabalhos aqui apresentados evidenciaram um efeito neuroprotetor de AR e AC por reduzir o estresse oxidativo, a ativação de vias inflamatórias e o dano genotóxico, envolvidos na progressão do processo epiléptico. / Antioxidants and anti-inflammatory compounds have been identified as an alternative to improve the treatment of epilepsy. The rosmarinic acid (RA) and its metabolite caffeic acid (CA) have their antioxidant and anti-inflammatory effects reported in several studies. Moreover, AR has inhibitory action on GABA transaminase (GABA-T), the enzyme that metabolizes an important neurotransmitter involved in epilepsy: GABA. In this thesis, we investigated the effect of RA and CA in seizure models and its neuroprotective action on oxidative stress, neuroinflammation and genotoxicity related to epilepsy. For this, first we used the kindling model induced by pentylenetetrazole (PTZ-induced kindling) for evaluating the effect of compounds on epileptogenesis. The compounds had no effect antiepileptogenic in vivo in this model, however, some doses of RA and CA showed a neuroprotective effect against oxidative stress and DNA damage induced by kindling in the cortex of the mice, demonstrating a beneficial effect against physiological changes related to epileptogenesis. Based on the fact of RA being an inhibitor of GABA-T, we evaluated the effect of compounds over potentiation of action of GABAergic agonist diazepam (DZP) on acute seizures induced by PTZ and pilocarpine (PIL) and in DZP-induced sleep test. The preventive effect of genotoxic damage induced by acute seizure models also was investigated. RA and CA showed potentiate GABAergic activation, which was evidenced by increase in latency to acute seizures PTZ-induced promoted by RA (4 mg/kg), and the reduction in latency to sleep in DZP-induced sleep test promoted by RA (4 mg/kg) and AC (8 mg/kg). In addition, RA and CA (4 mg/kg) reduced the genotoxic damage caused by PIL. Moreover, we found (in vitro) that the pre-treatment with RA was able to modulate microglial activation, producing a pattern of anti-inflammatory activation, reducing the formation of reactive species and the expression of proinflammatory cytokines, consequently reducing genotoxic damage induced by inflammation. These responses were related to the possible inhibition of apoptotic pathway by RA. In short, the works presented here showed a neuroprotective effect of RA and CA because of redution of oxidative stress, inflammatory pathways and genotoxic damage, involved in the progression of the epileptic process.

A hemorragia intracerebral (HIC) é uma das principais causas de mortalidade em todo mundo, desencadeando eventos adversos e déficits neurológicos graves. A partir disso, essa tese teve como objetivo avaliar as respostas celulares e suas interações envolvidas na HIC e investigar os possíveis mecanismos que contribuem para os déficits sensório-motores relacionados, principalmente, à habilidade dos membros anteriores dos animais. Para isso foram realizados dois experimentos. Em ambos os animais de 90 dias foram submetidos à HIC induzida por colagenase no estriado dorsolateral. No primeiro experimento, após 24h, 72h e 7 dias os animais foram avaliados funcionalmente, e histologicamente quanto ao volume da lesão. Ainda, durante 6h, 24h, 72h e 7 dias, o estriado e o córtex foram avaliados quanto ao número de células neuronais, gliais, e em apoptose; a expressão de GFAP e S100B, no estriado e no córtex, a secreção de S100B, no soro e líquido cérebro-espinhal (LCE). A HIC provocou déficits sensório-motores e perda tecidual; induziu morte neuronal e gliose reativa no estriado. O córtex obteve uma resposta astrocitária similar, mas quanto à microglia houve um aumento apenas em 6h. A S100B correlacionou-se positivamente com o número de células gliais; e negativamente com a força e função dos membros anteriores dos animais lesados. No segundo experimento, foram avaliados o estado oxidativo, o nível de TNF-α, a atividade da glutamina sintetase (GS) e a captação de glutamato nos tempos de 6h, 24h, 72h e 7 dias pós-lesão. Após 6h da HIC houve aumento no nível de TNF-α e excitotoxicidade glutamatérgica; entretanto, a função de clearence astrocitário foi restabelecida 7 dias após o dano. A HIC gerou estresse oxidativo e aumento de antioxidantes endógenos, além disso, as espécies reativas de oxigênio (ERO) inibiram a atividade da GS em 24h e 7 dias após a lesão. A partir dos resultados obtidos, podemos concluir que a S100B atua na lesão hemorrágica produzindo respostas distintas, região dependente, contribuindo para o aumento das células gliais no estriado e córtex, correlacionando-se positivamente com os déficits funcionais. Este é o primeiro relato descrevendo a relação da S100B com as alterações celulares e funcionais durante a progressão da HIC experimental. Além disso, os mecanismos envolvidos na HIC demonstraram padrões e respostas envolvendo excitotoxicidade glutamatérgica e inflamação, que colaboraram para o dano oxidativo; em resposta ao estresse houve o aumento de antioxidantes endógenos que podem ter contribuído para o reestabelecimento da função astrocitária. / Intracerebral haemorrhage (ICH) is a major cause of mortality worldwide, triggering adverse events and serious neurological deficits. From this standpoint, this thesis aimed to evaluate the cellular responses and interactions involved in ICH and investigate the possible mechanisms that contribute to sensory-motor deficits, mainly related to the ability of the forelimbs of animals. For this, two experiments were conducted. In both, animals of 90 days of age were subjected to ICH induced by collagenase in dorsolateral striatum. In the first experiment, after 24h, 72h and 7 days, the animals were evaluated functionally, and histologically about the lesion volume. Also, at 6h, 24h, 72h and 7 days, the striatum and the cortex was evaluated as the number of neuronal, glial and apoptotic cells; the expression of GFAP and S100B, the striatum and cortex, S100B secretion in serum and cerebrospinal fluid (CSF). ICH caused sensorimotor deficits and tissue loss; induced neuronal death and reactive gliosis in the striatum. The cortex astrocyte obtained a similar response, but about the microglia there was an increase only at 6h. S100B was positively correlated with the number of glial cells; and negatively correlated with the strength and function of the forelimbs of injured animals. In the second experiment, were evaluated the oxidative state, the level of TNF-α, the activity of glutamine synthetase (GS) and glutamate uptake at 6h, 24h, 72h and 7 days post-injury. After 6h from ICH, there was an increase in TNF-α levels and glutamatergic excitotoxicity; however, the astrocyte clearance function was established 7 days after the damage. HIC generated oxidative stress and increase of endogenous antioxidants, in addition, reactive oxygen species (ROS) inhibited the activity of GS in 24h and 7 days after injury. Based on the results obtained, we can conclude that S100B acts in hemorrhagic lesions producing different responses, region-dependent, contributing to the increase in glial cells in the striatum and cortex, correlating positively with functional deficits. This is the first report describing the relation of S100B with cellular and functional changes during the progression of experimental ICH. Furthermore, the mechanisms involved in ICH displayed patterns and responses involving glutamatergic excitotoxicity and inflammation, that contributed to oxidative damage; in response to stress there was an increase of endogenous antioxidants which may have contributed to the re-establishment of astrocyte function.