Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

by Mo Flora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 168-184). / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Stroke as a major disabling disease --- p.1 / Chapter 1.2 --- Classification of stroke --- p.4 / Chapter 1.3 --- Risk factors attributing to stroke --- p.15 / Chapter 1.4 --- Experimental methods to induce cerebral ischemia --- p.19 / Chapter 1.4.1 --- The establishment of animal models for stroke --- p.21 / Chapter 1.4.2 --- Gerbil as a putative model --- p.25 / Chapter 1.5 --- Mechanisms of focal ischemia damage --- p.30 / Chapter 1.6 --- Potential biochemical markers for cerebral ischemia --- p.38 / Chapter 1.7 --- Aim of investigation --- p.48 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Common chemicals --- p.49 / Chapter 2.2 --- Common bench solutions --- p.52 / Chapter 2.3 --- Animals / Chapter 2.3.1 --- Gerbils --- p.52 / Chapter 2.3.2 --- Rabbit --- p.53 / Chapter 2.4 --- Establishment of an animal model / Chapter 2.4.1 --- Surgical methods for common carotid artery (CCA) ligation --- p.54 / Chapter 2.5 --- Methods to determine stroke conditions of gerbils / Chapter 2.5.1 --- Ocular fundus examination --- p.56 / Chapter 2.5.2 --- Stroke index --- p.56 / Chapter 2.5.3 --- Inclined plane method --- p.59 / Chapter 2.6 --- Preparation of gerbil brain for subsequent analysis / Chapter 2.6.1 --- Preparation of gerbil brain slices --- p.61 / Chapter 2.6.2 --- "2,3,5-triphenytetrazolium chloride (TTC) for quantitative staining of brain slices" --- p.61 / Chapter 2.6.3 --- Preparation of normal and stroke gerbil brain extract --- p.62 / Chapter 2.7 --- Polyacrylamide gel electrophoresis (PAGE) using a discontinuous buffer system / Chapter 2.7.1 --- Stock reagents --- p.63 / Chapter 2.7.2 --- Separation gel preparation --- p.65 / Chapter 2.7.3 --- Stacking gel preparation --- p.66 / Chapter 2.7.4 --- Electrophoresis conditions --- p.67 / Chapter 2.7.5 --- Staining and destaining --- p.67 / Chapter 2.8 --- Two dimensional slab gel electrophoresis / Chapter 2.8.1 --- Equipment --- p.70 / Chapter 2.8.2 --- Chemical --- p.70 / Chapter 2.8.3 --- Procedure --- p.74 / Chapter 2.9 --- Production of rabbit polyclonal antibodies against isolated stroke protein / Chapter 2.9.1 --- Isolation of stroke protein band from SDS-PAGE slab gel --- p.78 / Chapter 2.9.2 --- Production of anti-stroke protein serum in rabbits --- p.79 / Chapter 2.10 --- Western blotting method / Chapter 2.10.1 --- Reagents --- p.80 / Chapter 2.10.2 --- Procedures --- p.81 / Chapter 2.11 --- Extraction of total cellular RNA by lithium chloride method / Chapter 2.11.1 --- Reagents --- p.83 / Chapter 2.11.2 --- Procedures --- p.84 / Chapter 2.11.3 --- Checking the purity of the extracted RNA --- p.85 / Chapter 2.12 --- Purification of mRNA / Chapter 2.12.1 --- Reagents --- p.85 / Chapter 2.12.2 --- Procedure --- p.86 / Chapter 2.13 --- Verification of purity of mRNA / Chapter 2.13.1 --- Reagents --- p.87 / Chapter 2.13.2 --- Procedure --- p.88 / Chapter 2.14 --- Translation of gerbil brain mRNA in reticulocyte lysates and analysis of its product by SDS PAGE / Chapter 2.14.1 --- Reagents --- p.89 / Chapter 2.14.2 --- Procedures --- p.89 / Chapter CHAPTER THREE --- ESTABLISHMENT OF AN ANIMAL STROKE MODEL / Chapter 3.1 --- Foreword --- p.92 / Chapter 3.2 --- Preliminary studies / Chapter 3.2.1 --- Introduction --- p.92 / Chapter 3.2.2 --- Results --- p.93 / Chapter 3.2.3 --- Discussion --- p.96 / Chapter 3.3 --- Survival rate analysis / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Result --- p.98 / Chapter 3.3.3 --- Discussion --- p.102 / Chapter 3.4 --- Neurologic signs of ischemia / Chapter 3.4.1 --- Introduction --- p.103 / Chapter 3.4.2 --- Result --- p.105 / Chapter 3.4.3 --- Discussion --- p.111 / Chapter 3.5 --- Ocular fundus examination / Chapter 3.5.1 --- Introduction --- p.112 / Chapter 3.5.2 --- Result --- p.114 / Chapter 3.5.3 --- Discussion --- p.116 / Chapter 3.6 --- Inclined plane method / Chapter 3.6.1 --- Introduction --- p.117 / Chapter 3.6.2 --- Result --- p.118 / Chapter 3.6.3 --- Discussion --- p.121 / Chapter 3.7 --- Histologic examination using TTC as staining agent / Chapter 3.7.1 --- Introduction --- p.122 / Chapter 3.7.2 --- Result --- p.124 / Chapter 3.7.3 --- Discussion --- p.129 / Chapter CHAPTER FOUR --- IDENTIFICATION OF ALTERED PROTEIN PATTERN IN THE - BRAINS OF STROKE GERBILS BY ELECTROPHORETIC METHODS / Chapter 4.1 --- Separation of soluble brain extracts by SDS-PAGE analysis / Chapter 4.1.1 --- Introduction --- p.130 / Chapter 4.1.2 --- Result --- p.132 / Chapter 4.1.3 --- Discussion --- p.140 / Chapter 4.2 --- Two dimensional electrophoretic analysis of soluble brain extracts from stroke gerbils / Chapter 4.2.1 --- Introduction --- p.142 / Chapter 4.2.2 --- Result --- p.143 / Chapter 4.2.3 --- Discussion --- p.148 / Chapter CHAPTER FIVE --- ISOLATION OF STROKE-ASSOCIATED PROTEIN FROM BRAINS OF STROKE GERBILS BY IMMUNOCHEMICAL METHOD / Chapter 5.1 --- Introduction --- p.149 / Chapter 5.2 --- Result --- p.151 / Chapter 5.3 --- Discussion --- p.153 / Chapter CHAPTER SIX --- DETECTION OF NEW PROTEIN TRANSLATED FROM MESSENGER RIBONUCLEIC ACID FROM BRAINS OF STROKE GERBIL / Chapter 6.1 --- Introduction / Chapter 6.1.1 --- Extraction of stroke gerbil brain messenger ribonucleic acid --- p.154 / Chapter 6.1.2 --- Translation of mRNA --- p.154 / Chapter 6.2 --- Results / Chapter 6.2.1 --- Yield of total cellular RNA --- p.157 / Chapter 6.2.2 --- Verification of purity of mRNA --- p.157 / Chapter 6.2.3 --- Autoradiographic patterns of translated proteins --- p.159 / Chapter 6.3 --- Discussion --- p.163 / Chapter CHAPTER SEVEN --- GENERAL DISCUSSION --- p.165 / BIBLIOGRAPHY --- p.168

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 12 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

Cerebral ischemia is the principal cause of morbidity and mortality worldwide. In addition to neuronal loss associated with hypoxic-ischemic damage, cerebral ischemia is characterized by a neuromicrovascular injury. Nitrative stress and lipid peroxidation increase in hypoxic-ischemic damages and play an essential role in neuromicrovascular injury leading to cerebral ischemia. We hypothesized that newly described lipid peroxidation products, termed trans-arachidonic acids (TAA), could be implicated in the pathogenesis of hypoxia-ischemia by affecting the cerebral vasomotricity and microvascular integrity. / The effects of TAA on neuromicrovascular tone were tested ex vivo by monitoring the changes in vascular diameter of rat cerebral pial microvessels. Four isomers of TAA, namely 5 E-AA, 8E-AA, IIE-AA and 14 E-AA induced an endothelium-dependent vasorelaxation. Possible mechanisms involved in TAA-induced vasorelaxation were thoroughly investigated. Collectively, data enclosed revealed that TAA induce cerebral vasorelaxation through the interactive activation of BKCa channels with heme oxygenase-2. This interaction leads to generation of carbon monoxide which in turn activates soluble guanylate cyclase and triggers vasorelaxation. / Chronic effects of TAA on microvascular integrity were examined by generating a unilateral hypoxic-ischemic (HI) model of cerebral ischemia on newborn rat pups. Our HI model showed microvascular degeneration as early as 24h post-HI, preceded by an increase in cerebral TAA levels. HI-induced microvascular lesions were dependent on nitric oxide synthase activation and ensued TAA formation. Although the molecular mechanisms leading to TAA-induced microvascular degeneration were, in part uncovered for the retina, the primary site of action of TAA remains unknown. We demonstrated that TAA binds and activates GPR40 receptor, a newly described free fatty acid receptor. Importantly, GPR40 receptor knock-out prevents TAA-induced reduction in cerebral microvascular density and limits HI-induced brain infarct.

No description available.

A perda da permeabilidade seletiva de membrana celular causada pela isquemia leva ao acúmulo de sódio e edema miocárdico. Este fenômeno tem implicações importantes na estrutura e função do ventrículo esquerdo, nas primeiras horas após infarto do miocárdio. Objetivou-se investigar a hipótese de que, durante as primeiras horas após oclusão coronária prolongada e restabelecimento de fluxo completo, a taxa de acúmulo de sódio miocárdico é determinado pela integridade da microvasculatura. Utilizou-se imagem de ressonância magnética do sódio-23 em 3 dimensões, para monitorizar as alterações do conteúdo de sódio miocárdico no tempo, em um modelo canino in vivo e com tórax fechado (n = 19) de infarto do miocárdio e reperfusão. Seis animais apresentaram fibrilação ventricular durante a oclusão ou imediatamente após reperfusão coronária, não completando o protocolo. Em quatro experimentos não se detectou nenhuma área de infarto, por nenhum dos métodos utilizados. Um animal foi submetido a oclusão coronária permanente. Os oito animais restantes constituíram o grupo de infartos reperfundidos. Destes, infartos com obstrução microvascular (n = 4), detectados por microesfera radioativa e por imagem por ressonância magnética do hidrogênio realçada com contraste, mostraram uma taxa menor de acúmulo de sódio, assim como um menor fluxo sangüíneo 20 minutos e 6 horas após reperfusão. A ausência de obstrução microvascular nos infartos (n = 4) esteve associada a taxas maiores de acúmulo do sódio e maior restabelecimento do fluxo sangüíneo miocárdico. Além disso, o tamanho do infarto por imagem por ressonância magnética do sódio-23 apresentou boa correlação com o tamanho do infarto pela anatomopatologia (cloreto de trifeniltetrazólio ou TTC) e pela imagem de ressonância do hidrogênio realçada por contraste (hiperintensificação ou realce tardio) 9 horas após reperfusão. Conclui-se que, em infartos do miocárdio reperfundidos, o acúmulo de sódio é dependente da integridade microvascular e está diminuído em regiões de obstrução microvascular, comparado com regiões miocárdicas com microvasculatura patente. A imagem por ressonância do sódio-23 pode ser um instrumento útil para a monitorização in vivo do conteúdo do sódio no infarto agudo do miocárdio. / Loss of membrane permeability caused by ischemia leads to cellular sodium accumulation and myocardial edema. This phenomenon has important implications to left ventricular structure and function in the first hours after myocardial infarction. We hypothesized that during this period of time, after prolonged coronary occlusion and complete reflow, the rate of myocardial sodium accumulation is governed by microvascular integrity. We used 3-dimensional 23 Na magnetic resonance imaging to monitor myocardial sodium content changes over time in an in vivo closed-chest canine model (n = 19) of myocardial infarction and reperfusion. Six animals had ventricular fibrillation during occlusion or immediately after coronary reperfusion, and did not finish the protocol. Myocardial infarction was not detected in four experiments, by any of utilized methods. In one experiment, we produced permanent coronary occlusion. The remaining eight animals constituted the reperfused myocardial infarction group. From those, infarcts with microvascular obstruction n = 4) defined by both radioactive microsphere and contrast-enhanced 1 H magnetic resonance imaging showed a slower rate of sodium accumulation as well as lower blood flow at 20 minutes and 6 hours after reperfusion. Conversely, the absence of microvascular obstruction (n = 4) was associated with faster rates of sodium accumulation and greater blood flow restoration. In addition, infarct size by 23 Na magnetic resonance imaging correlated best with infarct size by triphenyltetrazolium chloride and contrast-enhanced 1 H magnetic resonance imaging at 9 hours after reperfusion. We conclude that in reperfused myocardial infarction, sodium accumulation is dependent on microvascular integrity and is slower in regions of microvascular obstruction compared with those with patent microvasculature. Finally, 23 Na magnetic resonance imaging can be a useful tool for monitoring in vivo myocardial sodium content in acute myocardial infarction.

A perda da permeabilidade seletiva de membrana celular causada pela isquemia leva ao acúmulo de sódio e edema miocárdico. Este fenômeno tem implicações importantes na estrutura e função do ventrículo esquerdo, nas primeiras horas após infarto do miocárdio. Objetivou-se investigar a hipótese de que, durante as primeiras horas após oclusão coronária prolongada e restabelecimento de fluxo completo, a taxa de acúmulo de sódio miocárdico é determinado pela integridade da microvasculatura. Utilizou-se imagem de ressonância magnética do sódio-23 em 3 dimensões, para monitorizar as alterações do conteúdo de sódio miocárdico no tempo, em um modelo canino in vivo e com tórax fechado (n = 19) de infarto do miocárdio e reperfusão. Seis animais apresentaram fibrilação ventricular durante a oclusão ou imediatamente após reperfusão coronária, não completando o protocolo. Em quatro experimentos não se detectou nenhuma área de infarto, por nenhum dos métodos utilizados. Um animal foi submetido a oclusão coronária permanente. Os oito animais restantes constituíram o grupo de infartos reperfundidos. Destes, infartos com obstrução microvascular (n = 4), detectados por microesfera radioativa e por imagem por ressonância magnética do hidrogênio realçada com contraste, mostraram uma taxa menor de acúmulo de sódio, assim como um menor fluxo sangüíneo 20 minutos e 6 horas após reperfusão. A ausência de obstrução microvascular nos infartos (n = 4) esteve associada a taxas maiores de acúmulo do sódio e maior restabelecimento do fluxo sangüíneo miocárdico. Além disso, o tamanho do infarto por imagem por ressonância magnética do sódio-23 apresentou boa correlação com o tamanho do infarto pela anatomopatologia (cloreto de trifeniltetrazólio ou TTC) e pela imagem de ressonância do hidrogênio realçada por contraste (hiperintensificação ou realce tardio) 9 horas após reperfusão. Conclui-se que, em infartos do miocárdio reperfundidos, o acúmulo de sódio é dependente da integridade microvascular e está diminuído em regiões de obstrução microvascular, comparado com regiões miocárdicas com microvasculatura patente. A imagem por ressonância do sódio-23 pode ser um instrumento útil para a monitorização in vivo do conteúdo do sódio no infarto agudo do miocárdio. / Loss of membrane permeability caused by ischemia leads to cellular sodium accumulation and myocardial edema. This phenomenon has important implications to left ventricular structure and function in the first hours after myocardial infarction. We hypothesized that during this period of time, after prolonged coronary occlusion and complete reflow, the rate of myocardial sodium accumulation is governed by microvascular integrity. We used 3-dimensional 23 Na magnetic resonance imaging to monitor myocardial sodium content changes over time in an in vivo closed-chest canine model (n = 19) of myocardial infarction and reperfusion. Six animals had ventricular fibrillation during occlusion or immediately after coronary reperfusion, and did not finish the protocol. Myocardial infarction was not detected in four experiments, by any of utilized methods. In one experiment, we produced permanent coronary occlusion. The remaining eight animals constituted the reperfused myocardial infarction group. From those, infarcts with microvascular obstruction n = 4) defined by both radioactive microsphere and contrast-enhanced 1 H magnetic resonance imaging showed a slower rate of sodium accumulation as well as lower blood flow at 20 minutes and 6 hours after reperfusion. Conversely, the absence of microvascular obstruction (n = 4) was associated with faster rates of sodium accumulation and greater blood flow restoration. In addition, infarct size by 23 Na magnetic resonance imaging correlated best with infarct size by triphenyltetrazolium chloride and contrast-enhanced 1 H magnetic resonance imaging at 9 hours after reperfusion. We conclude that in reperfused myocardial infarction, sodium accumulation is dependent on microvascular integrity and is slower in regions of microvascular obstruction compared with those with patent microvasculature. Finally, 23 Na magnetic resonance imaging can be a useful tool for monitoring in vivo myocardial sodium content in acute myocardial infarction.