Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.

Introdução: A isquemia cerebral é uma desordem da função cerebral ocasionado pela supressão sanguínea no tecido cerebral sem nenhuma outra causa aparente do que a vascular. Estudos revelam os danos causados pela isquemia cerebral focal repercutem não apenas na região da lesão isquêmica, mas também em outras regiões do encéfalo, dentre elas o cerebelo. O etanol atua diminuindo o tempo de reação do corpo e a resposta reflexa, produzindo até mesmo perda de coordenação motora. Por tempos, estudos tem verificado a ação do etanol no cerebelo. Objetivos: Este trabalho tem por objetivo analisar o córtex cerebelar de ratos submetidos a um modelo experimental de isquemia cerebral focal transitória por oclusão da ACM durante 90 minutos, seguida por reperfusão de 48 horas, associado ou não a modelo de alcoolismo. Material e métodos: Foram utilizados 50 ratos Wistar adultos, subdivididos em 5 grupos experimentais: grupo controle (C): animais submetidos apenas à anestesia; grupo sham (S): animais submetidos à simulação completa do procedimento cirúrgico; grupo isquêmico (I): animais submetidos à isquemia cerebral focal por 90 minutos seguido por reperfusão de 48 horas; grupo alcoolizado (A): animais que receberam diariamente álcool etílico absoluto diluído a 20% em água durante quatro semanas; e, grupo isquêmico e alcoolizado (IA): animais submetidos ao mesmo tratamento do grupo A e que, após quatro semanas foram submetidos à isquemia cerebral focal durante 90 minutos, seguido por reperfusão de 48 horas. As amostras do cerebelo coletadas e realizado a análise de imunohistoquímica da proteína CASPASE-9 e a análise sérica por meio de PCR - RT dos miRNAS miR-21, miR-126 e o miR155. Resultados: A expressão de CASPASE 9 teve maior expressão no grupos I, A e I+A. A análise dos miRNAS, o miR-126 foi maior nos grupos A e I+A, o miR-155 foi maior nos grupos I e I+A. Conclusões: Podemos concluir que a ocorrência de apoptose no córtex cerebelar, mesmo distante do foco isquêmico e ques miRNAs 126 e 155 apresentam correlação com a apoptose celular em ratos isquêmicos e submetidos ao modelo de alcoolismo crônico / INTRODUCTION: Cerebral ischemia is a disorder of brain function caused by blood suppression in brain tissue with no apparent cause other than vascular. Studies reveal the damage caused by focal cerebral ischemia to affect not only the region of the ischemic lesion but also other regions of the brain, including the cerebellum. Ethanol acts by decreasing the reaction time of the body and the reflex response, producing even loss of motor coordination. For some time, studies have verified the action of ethanol in the cerebellum. AIMS: This study aims to analyze the cerebellar cortex of rats submitted to an experimental model of transient focal cerebral ischemia by ACM occlusion for 90 minutes, followed by reperfusion of 48 hours, associated or not with the model of alcoholism. METHODS: Fifty adult Wistar rats were used, subdivided into 5 experimental groups: control group (C): animals submitted to anesthesia only; sham group (S): animals submitted to complete simulation of the surgical procedure; ischemic group (I): animals submitted to focal cerebral ischemia for 90 minutes followed by reperfusion of 48 hours; alcoholic group (A): animals that received daily absolute ethanol diluted 20% in water for four weeks; and ischemic and alcoholized group (AI): animals submitted to the same treatment as group A and after four weeks were submitted to focal cerebral ischemia for 90 minutes, followed by reperfusion of 48 hours. The cerebellum samples were collected and the immunohistochemical analysis of the CASPASE-9 protein and the serum analysis by means of RT-PCR of miRNAS miR-21, miR-126 and miR155 were performed. RESULTS: The expression of CASPASE 9 had higher expression in groups I, A and I + A. The miRNAS analysis, miR-126 was higher in groups A and I + A, miR-155 was higher in groups I and I + A. CONCLUSIONS: We can conclude that apoptosis occurs in the cerebellar cortex, even if it is distant from the ischemic focus, and that miRNAs 126 and 155 present a correlation with cellular apoptosis in ischemic rats and submitted to the chronic alcohol model

Stroke is a leading cause of neurological disability, often resulting in long-term motor impairments due to damage to the striatum and/or motor cortex. While both humans and animals show spontaneous recovery following stroke, little is known about how the injury location affects recovery and what causes recovery to plateau. This information is essential in order to improve current rehabilitation practice and develop new therapies to enhance recovery. In this thesis, we used endothelin-1 (ET-1), a potent vasoconstrictor, to produce focal infarcts in the forelimb motor cortex (FMC), the dorsolateral striatum (DLS) or both the FMC and DLS in male Sprague-Dawley rats. In the first experiment, the spontaneous recovery profile of animals was followed over an 8-week period using multiple behavioural tasks assessing motor function and limb preference to identify how recovery varies depending on injury location. Infarct volumes were measured to determine the association between injury and behavioural outcome. All three groups had significant functional impairments on the Montoya staircase, beam traversal, and cylinder tests following stroke, with the combined group having the largest and most persistent impairments. Importantly, spontaneous recovery was not simply dependent on lesion volume but on the lesion location and the behavioural test employed. In the second experiment, we focused on a potential cellular mechanism thought to underlie post-stroke plasticity and functional recovery. In a separate cohort of animals, we assessed how plasticity-limiting perineuronal nets (PNNs) and associated parvalbumin-positive (PV) GABAergic interneurons change following similar ET-1 strokes as in the prior experiment. A significant reduction in the density of PNNs was observed in the perilesional cortex of animals that received a cortical-only or combined stroke but not a striatal-only injury. Although there were no significant differences in the density of PV interneurons between sham and stroked groups, a significant negative correlation existed between cortical infarct volume and the density of PV interneurons in the perilesional cortex. Taken together these results demonstrate that lesion location influences motor recovery and neuroplastic changes following stroke. This supports the idea that a “one size fits all” approach for stroke rehabilitation may not be effective and treatment needs to be individualized to the patient.

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Hypoxic-ischemic insult is a most common form of perinatal brain damage that threatens a newborn's life and can leads to permanent neurological sequelae. However, detailed aspects of the cerebral ischemia in the immature brain stay unanswered. We decide to use the model of focal cerebral ischemia induced by intrahippocampal endothelin-1 (ET-1) in 12-days-old rats. The knowledge about consequences of ET-1 induced ischemia and the role of endothelin receptors (ETA and ETB) in ischemia-induced consequences in immature brain are poor at present. Agonists and selective antagonists of the ETA and ETB receptors were used to determine the role of these receptors in the development of ischemia, changes in regional blood flow and tissue oxygenation, local changes of biochemical parameters and acute neuronal death. Our results indicates, that activation of the ETA receptors causes a strong decrease of the blood flow, induced related hypoxia and subsequent neuronal degeneration, whereas activation of ETB receptors has likely modulatory role. Moreover, ischemia causes increase of excitatory amino acids concentration, whereas inhibitory amino acid, except taurine, decreased after ischemia. These facts provides new insights in a case of perinatal ischemia. This thesis demonstrates the wide range of different effects of...