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Angiogenesis in cerebral ischemia border zone
Nulíčková, Radka ; Otáhal, Jakub (advisor) ; Brime, Tufikameni (referee)
Title of work: Angiogenesis in the border zones of cerebral ischemia The aims of work: The aim of this diploma thesis was to determinate the parameter of the angiogenesis after focal brain ischemia induced by photothrombosis in P7 rat pups. This thesis should answer the question, if there is some difference in vascular density according to the distance from the lesion and if there is some diference between the vascular density in normal brain and the brain with the lesion. Materials and Methods: Cerebral ischemia was induced in P7 rat pups with photothombic method. Intravenous administration of bengal rose dye was followed by illumination of beam of green laser over the cortex for 5 minutes at 0,5s on/off cycles. Bengal rose dye is potent photosensitive substance and the ilumination resulted in agregation of trombocytes and thrombosis. Cerebral ischemia evolved due to the thrombosis. After two months, the animals were overdosed with urethane and transcardially perfused. Then thein brain were cryoprotected and then cut into 50µm slices and prepaired for histological staining. The stained slices were the investigated under mikroskope. The microvessels in the cortex were counted and the density was estimated by stereological method. Results: Two brain with ischemic lesion and two control brain were...
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The effect of the canonical Wnt singalling pathway on the differentiation of polydendrocytes after ischemic brain injury
Knotek, Tomáš ; Anděrová, Miroslava (advisor) ; Romanyuk, Natalyia (referee)
Polydendrocytes, or NG2 glia, are fourth type of glial cells in mammal central nervous system. In the adult brain, NG2 glia represent important cell type with respect to their role in gliogenesis and nervous tissue regeneration following injury. Ligands from the Wingless/Int (Wnt) family play key role in proliferation and differentiation of NG2 glia and they can also influence regeneration of nervous tissue after ischemia. The aim of this thesis was to elucidate the role of NG2 glia in neurogenesis and gliogenesis following ischemic brain injury and investigate the impact of Wnt signalling on the reaction of NG2 glia to this type of injury. To fulfil these aims, transgenic mouse strains with tamoxifen-inducible recombination, that enabled simultaneous expression of red fluorescent dye and either activation or inhibition of the Wnt signalling pathway in NG2 glia, were employed. To induce ischemic injury, middle cerebral artery occlusion model was used. Changes in differentiation and electrophysiological properties of NG2 glia were analysed using patch-clamp technique. Activation of the Wnt signalling pathway under physiological conditions and 7 days after ischemic injury led to increased differentiation of NG2 glia toward astrocytes, while 3 days after ischemic injury activation of this signalling...
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Acute neuroinflammatory reaction after focal cerebral ischemia
Ambrož, Ondřej ; Otáhal, Jakub (advisor) ; Konopková, Renata (referee)
Title: Acute neuroinflammatory reaction after focal cerebral ischemia Aim: The aim of this thesis is to evaluate neuroinflammatory response after focal cerebral cortical ischemia. Also, familiarizing with the method of displaying damage of blood brain barriers, neurons and the possibility of detection of microglia cells as a marker of acute neuroinflammatory processes. Methodology: This is an experimental study. We brought about cortical cerebral ischemia in rats using an application of photosensitive dye "bengal red," and a green laser. Two animals were were given the additional application of "Evans blue" in order to visualize the defects of the blood brain barrier. The animals were returned to their cage for the time needed before they were induced terminal anesthesia. This was followed by the process of brain perfusion, slicing the brain in sections 50 µm thick and then applied these sections onto slides. Sections with applied EB were immediately analyzed under the microscope. Sections to illustrate neuronal death were immunohistochemically stained via the Nissl method. Sections visualizing microglial activity were stained using CD11b antibody. Results: Following the induction of focal ischemia there occured brain tissue damage. In the vicinity of lesion there is degeneration of neurons and...
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The effect of the canonical Wnt singalling pathway on the differentiation of polydendrocytes after ischemic brain injury
Knotek, Tomáš ; Anděrová, Miroslava (advisor) ; Romanyuk, Natalyia (referee)
Polydendrocytes, or NG2 glia, are fourth type of glial cells in mammal central nervous system. In the adult brain, NG2 glia represent important cell type with respect to their role in gliogenesis and nervous tissue regeneration following injury. Ligands from the Wingless/Int (Wnt) family play key role in proliferation and differentiation of NG2 glia and they can also influence regeneration of nervous tissue after ischemia. The aim of this thesis was to elucidate the role of NG2 glia in neurogenesis and gliogenesis following ischemic brain injury and investigate the impact of Wnt signalling on the reaction of NG2 glia to this type of injury. To fulfil these aims, transgenic mouse strains with tamoxifen-inducible recombination, that enabled simultaneous expression of red fluorescent dye and either activation or inhibition of the Wnt signalling pathway in NG2 glia, were employed. To induce ischemic injury, middle cerebral artery occlusion model was used. Changes in differentiation and electrophysiological properties of NG2 glia were analysed using patch-clamp technique. Activation of the Wnt signalling pathway under physiological conditions and 7 days after ischemic injury led to increased differentiation of NG2 glia toward astrocytes, while 3 days after ischemic injury activation of this signalling...
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Proliferation and differentiation of NG2-glia following ischemic brain injuries
Kirdajová, Denisa ; Anděrová, Miroslava (advisor) ; Machová Urdzíková, Lucia (referee)
NG2-glia, a fourth major glial cell population, were shown to posses wide proliferation and differentiation potential in vitro and in vivo, therefore the aim of this study was to compare the rate of proliferation and differentiation potential of NG2-glia after different types of brain injuries, such as global and focal cerebral ischemia (GCI, FCI) or stab wound (SW), as well as during aging. Moreover, we aimed to determine the role of Sonic hedgehog (Shh) in NG2-glia proliferation/differentiation after FCI. We used transgenic mice, in which tamoxifen triggers the expression of red fluorescent protein (tdTomato) in NG2-glia and cells derived therefrom. Proliferation and differentiation potential of tdTomato+ cells in sham operated animals (controls) and those after injury were determined by immunohistochemistry employing antibodies against proliferating cell nuclear antigen and glial fibrillary acidic protein. FCI was induced by middle cerebral artery occlusion, GCI by carotid occlusion with hypotension and SW by sagittal cortical cut. Shh signaling in vivo was activated or inhibited by Smoothened agonist or Cyclopamine, respectively. Compared to controls, the proliferation rate of tdTomato+ cells was increased after all types of injuries, while it declined in aged mice (15-18- months-old) after...
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Acute neuroinflammatory reaction after focal cerebral ischemia
Ambrož, Ondřej ; Otáhal, Jakub (advisor) ; Konopková, Renata (referee)
Title: Acute neuroinflammatory reaction after focal cerebral ischemia Aim: The aim of this thesis is to evaluate neuroinflammatory response after focal cerebral cortical ischemia. Also, familiarizing with the method of displaying damage of blood brain barriers, neurons and the possibility of detection of microglia cells as a marker of acute neuroinflammatory processes. Methodology: This is an experimental study. We brought about cortical cerebral ischemia in rats using an application of photosensitive dye "bengal red," and a green laser. Two animals were were given the additional application of "Evans blue" in order to visualize the defects of the blood brain barrier. The animals were returned to their cage for the time needed before they were induced terminal anesthesia. This was followed by the process of brain perfusion, slicing the brain in sections 50 µm thick and then applied these sections onto slides. Sections with applied EB were immediately analyzed under the microscope. Sections to illustrate neuronal death were immunohistochemically stained via the Nissl method. Sections visualizing microglial activity were stained using CD11b antibody. Results: Following the induction of focal ischemia there occured brain tissue damage. In the vicinity of lesion there is degeneration of neurons and...
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Angiogenesis in cerebral ischemia border zone
Nulíčková, Radka ; Otáhal, Jakub (advisor) ; Brime, Tufikameni (referee)
Title of work: Angiogenesis in the border zones of cerebral ischemia The aims of work: The aim of this diploma thesis was to determinate the parameter of the angiogenesis after focal brain ischemia induced by photothrombosis in P7 rat pups. This thesis should answer the question, if there is some difference in vascular density according to the distance from the lesion and if there is some diference between the vascular density in normal brain and the brain with the lesion. Materials and Methods: Cerebral ischemia was induced in P7 rat pups with photothombic method. Intravenous administration of bengal rose dye was followed by illumination of beam of green laser over the cortex for 5 minutes at 0,5s on/off cycles. Bengal rose dye is potent photosensitive substance and the ilumination resulted in agregation of trombocytes and thrombosis. Cerebral ischemia evolved due to the thrombosis. After two months, the animals were overdosed with urethane and transcardially perfused. Then thein brain were cryoprotected and then cut into 50µm slices and prepaired for histological staining. The stained slices were the investigated under mikroskope. The microvessels in the cortex were counted and the density was estimated by stereological method. Results: Two brain with ischemic lesion and two control brain were...
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