Yeung Ho Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 93-107). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Glucose Transport through the Blood Brain Barrier --- p.1 / Chapter 1.2 --- Roles of Astrocytes in the Brain --- p.4 / Chapter 1.3 --- Glucose Metabolism in Astrocytes --- p.8 / Chapter 1.4 --- Diseases Associated with Reduced Glucose Transport --- p.10 / Chapter 1.5 --- Extracellular Accumulation of Glutamate as a Cause for Epilepsy --- p.13 / Chapter 1.6 --- Regulations of Astrocyte-mediated Glutamate Uptake --- p.17 / Chapter 1.7 --- Aim and Hypothesis of the Project --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Primary Rat Astrocytes --- p.24 / Chapter 2.1.2 --- Cell Culture Materials --- p.24 / Chapter 2.1.3 --- Chemicals --- p.26 / Chapter 2.1.4 --- Reagents for the Determination of Gene Expressions --- p.26 / Chapter 2.1.5 --- Reagents for the Determination of Protein Expressions --- p.29 / Chapter 2.1.6 --- Reagents for Functional Assays --- p.33 / Chapter 2.1.6.1 --- Reagents for Enzyme-Linked Immunosorbent Assay (ELISA) of IL-6 --- p.33 / Chapter 2.1.6.2 --- Reagents for Glutamate Uptake Assay --- p.33 / Chapter 2.1.6.3 --- Reagents for Extracellular Glutamate Determination Assay --- p.33 / Chapter 2.1.6.4 --- Reagents for Glucose Uptake Assay --- p.34 / Chapter 2.1.6.5 --- Reagents for MTT Assay --- p.34 / Chapter 2.1.6.6 --- Reagents for GFAP Immunostaining --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Preparation of Primary Astrocytes --- p.36 / Chapter 2.2.2 --- Determination of Gene Expressions by Reverse Transcription-Polymersase Chain Reaction (RT-PCR) --- p.37 / Chapter 2.2.3 --- Determination of Protein Expressions by Western Blotting --- p.40 / Chapter 2.2.4 --- ELISA --- p.43 / Chapter 2.2.5 --- Glutamate Uptake Assay --- p.44 / Chapter 2.2.6 --- Extracellular Glutamate Determination Assay --- p.44 / Chapter 2.2.7 --- Glucose Uptake --- p.45 / Chapter 2.2.8 --- MTT Assay --- p.46 / Chapter 2.2.9 --- GFAP Immunostaining --- p.46 / Chapter 2.2.10 --- Band Intensity Quantification --- p.47 / Chapter 2.2.11 --- Statistical Analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.49 / Chapter 3.1 --- Preparation of Primary Astrocyte Culture --- p.49 / Chapter 3.2 --- Effects of Glucose Deficiency on Astrocyte-mediated Glutamate Uptake --- p.51 / Chapter 3.2.1 --- Effects of Glucose Deficiency on the Expressions of Glutamate Transporters --- p.51 / Chapter 3.2.2 --- Effects of Glucose Deficiency on Glutamate Uptake in Primary Astrocytes --- p.56 / Chapter 3.3 --- Astrocytic Glucose Transport under Glucose Deficiency --- p.61 / Chapter 3.3.1 --- Effects of Glucose Deficiency on the Expressions and Secretion of Inflammatory Cytokines --- p.64 / Chapter 3.3.2 --- Effects of Exogenous Interleukin-6 on Energy Availability in Primary Astrocytes upon Glucose Deficiency --- p.70 / Chapter 3.4 --- Signaling Mechanism Mediating the Astrocytic Responses under Glucose Deficiency --- p.74 / Chapter 3.4.1 --- Effects of Glucose Deficiency on the Expressions of Total and Phosphorylated ERK1/2 in Primary Astrocytes --- p.74 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS --- p.81 / Chapter 4.1 --- Role of Astrocytes in Preventing Glutamate Excitotoxicity under Glucose Deficiency --- p.81 / Chapter 4.1.1 --- Neonatal Astrocytes as the Cell Model for Chronic Glucose Deficiency --- p.81 / Chapter 4.1.2 --- Effects of Glucose Deficiency on the Expressions of Glutamate Transporters and Glutamate Uptake --- p.83 / Chapter 4.1.3 --- Cytokines: Mediators for Energy Production in Astrocytes --- p.85 / Chapter 4.1.4 --- Summary of the Roles of Astrocyets under Prolonged Glucose Deficiency --- p.88 / Chapter 4.2 --- Establishment of an in vitro GlutlDS model --- p.89 / Chapter 4.3 --- Future Directions of the Project --- p.90 / Chapter 4.4 --- Conclusion --- p.92 / REFERENCES --- p.93 / APPENDIX --- p.108

Lai, Ching Janice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 148-168). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Nervous System and the Blood-Brain-Barrier --- p.3 / Chapter 1.3 --- Glial cells --- p.3 / Chapter 1.4 --- Studying Astrocyte Responses As a New Direction in Neuroscience --- p.4 / Chapter 1.5 --- The Roles of Astrocyte in the CNS --- p.5 / Chapter 1.5.1 --- Energy-Dependent Communication Between Neurons and Astrocytes --- p.7 / Chapter 1.5.2 --- Strategies for Metabolic Exchange Between Astrocytes and Neurons --- p.8 / Chapter 1.5.2.1 --- Provision of Energy Metabolites to Neurons by Astrocytes --- p.9 / Chapter 1.5.2.2 --- Glucose Transporters in the CNS --- p.10 / Chapter 1.5.2.3 --- The Lactate Shuttle Hypothesis --- p.12 / Chapter 1.5.2.4 --- The Regulation of Glucose Uptake at the Blood-Brain-Barrier (BBB) by the Activity of Neurons --- p.14 / Chapter 1.5.3 --- Alternation of Energy Metabolism in Neuropathy --- p.15 / Chapter 1.5.3.1 --- Ketone Body Shuttle Hypothesis --- p.15 / Chapter 1.5.3.2 --- The Utilization of Free Fatty Acids by the Brain --- p.17 / Chapter 1.5.4 --- The Provision of Neurotrophic Factors to Neurons by Astrocytes --- p.17 / Chapter 1.5.4.1 --- Neurotrophins --- p.18 / Chapter 1.5.4.1.1 --- Relationship Between Neurotrophins and Glucose --- p.20 / Chapter 1.5.4.2 --- S100B --- p.21 / Chapter 1.5.5 --- Astrocytic Cholesterol in Astrocytes as a Neurotrophic Factor --- p.22 / Chapter 1.6 --- Neuroprotective Effect of Glucose vi - --- p.23 / Chapter 1.7 --- Diseases Associated with Decreased Glucose Transport at the BBB --- p.24 / Chapter 1.7.1 --- Glucose Transporter Type 1 Deficiency Syndrome (GlutlDS) --- p.24 / Chapter 1.7.2 --- Hypoglycemia with Insulin Therapy for Diabetes Patients --- p.24 / Chapter 1.8 --- Aims and Hypothesis of Study --- p.26 / Chapter Chapter 2. --- 2 Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Cell Culture --- p.27 / Chapter 2.1.1.1 --- Cells --- p.27 / Chapter 2.1.1.1.1 --- C6 cells --- p.27 / Chapter 2.1.1.1.2 --- Primary Astrocytes --- p.27 / Chapter 2.1.1.2 --- Cell Culture Reagent --- p.27 / Chapter 2.1.2 --- Study of Growth Properties --- p.31 / Chapter 2.1.2.1 --- Equipment for Growth Curve Construction --- p.31 / Chapter 2.1.2.2 --- Reagents for Flow Cytometry --- p.32 / Chapter 2.1.2.3 --- Reagents for 3H-thymidine Incorporation Assay --- p.32 / Chapter 2.1.3 --- Study of Neurotrophic Properties --- p.33 / Chapter 2.1.3.1 --- Determination of Neurotrophic Factor Productions --- p.33 / Chapter 2.1.3.1.1 --- Reagents and Buffers for Northern Blot Analysis --- p.33 / Chapter 2.1.3.2 --- Reagents and Buffers for Western Blot Analysis --- p.43 / Chapter 2.1.3.2.1 --- Protein Assay --- p.43 / Chapter 2.1.3.2.2 --- Reagents for SDS Polyacrylamide Electrophoresis of Proteins --- p.44 / Chapter 2.1.3.2.3 --- Reagents for the Transfer of Protein to Membrane and Signal Detection --- p.47 / Chapter 2.1.4 --- Study of Lipid in Glial cells --- p.50 / Chapter 2.1.4.1 --- Determination of Genes Expression in Lipid Metabolism --- p.50 / Chapter 2.1.4.2 --- Reagents for Determination of Cholesterol and Fatty Acid Levels by Gas Chromatography --- p.50 / Chapter 2.2 --- Methods --- p.54 / Chapter 2.2.1 --- Cell culture --- p.54 / Chapter 2.2.1.1 --- Maintenance of C6 cells --- p.54 / Chapter 2.2.1.2 --- Primary Culture of Rat Astrocytes --- p.54 / Chapter 2.2.2 --- Study of Growth Properties of Glial Cells vii - --- p.56 / Chapter 2.2.2.1 --- Construction of cell growth curve --- p.56 / Chapter 2.2.2.2 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.56 / Chapter 2.2.2.3 --- Measurement of DNA Synthesis --- p.57 / Chapter 2.2.3 --- Study of Neurotrophic Properties --- p.58 / Chapter 2.2.3.1 --- Determination of Neurotrophic Facotor Production --- p.58 / Chapter 2.2.3.1.1 --- Northern Blot Analysis --- p.58 / Chapter 2.2.3.1.2 --- Western Blot Analysis --- p.67 / Chapter 2.2.3.2 --- Determination of Gene Expression in Lipid Metabolism --- p.72 / Chapter 2.2.3.2.1 --- Northern Blot Analysis --- p.72 / Chapter 2.2.3.2.2 --- RT-PCR --- p.72 / Chapter 2.2.3.3 --- Study of Lipid Profiles in Glial Cells --- p.73 / Chapter 2.2.3.3.1 --- Sample preparation --- p.73 / Chapter 2.2.3.3.2 --- Total Cholesterol Determination --- p.73 / Chapter 2.2.3.3.3 --- Total Fatty Acid Determination --- p.75 / Chapter 2.2.3.3.4 --- Quantification of Proteins --- p.76 / Chapter 2.2.4 --- Statistical Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- The effects of glucose deficiency on cell proliferation --- p.78 / Chapter 3.1.1 --- Direct Cell Count Assay --- p.78 / Chapter 3.1.2 --- Flow Cytometry Assay --- p.83 / Chapter 3.1.3 --- 3H-Thymidine Uptake Assay --- p.85 / Chapter 3.2 --- The Effects of Glucose Deficiency on Neurotrophic Properties of Glial Cells --- p.87 / Chapter 3.2.1 --- The Effects of Glucose Deficiency on mRNA and Protein Expressions of Neurotrophins --- p.88 / Chapter 3.2.1.1 --- Northern Blot Assays --- p.88 / Chapter 3.2.1.2 --- Western Blot Assays --- p.93 / Chapter 3.2.2 --- The Effects of Glucose Deficiency on Lipid Homeostasis --- p.96 / Chapter 3.2.2.1 --- Northern Blot Assays --- p.96 / Chapter 3.2.2.2 --- Gas Chromatography Assays --- p.101 / Chapter 3.2.2.2.1 --- Cholesterol Analyses --- p.102 / Chapter 3.2.2.2.2 --- Fatty Acid Analyses --- p.105 / Chapter Chapter 4 --- Discussion --- p.115 / Chapter 4.1 --- The in vitro Model of Hypoglycorrhachia --- p.115 / Chapter 4.2 --- Decreased Glucose Level Triggers Changes of Gial Cells Proliferation --- p.117 / Chapter 4.3 --- Expression of Neurotrophic Factor under Glucose Deficiency viii - --- p.120 / Chapter 4.3.1 --- Alteration of the Expression of Neurotrophins --- p.120 / Chapter 4.3.1.1 --- NGF --- p.122 / Chapter 4.3.1.2 --- BDNF --- p.123 / Chapter 4.3.1.3 --- NT-3 --- p.126 / Chapter 4.3.1.4 --- NT-4/5 --- p.128 / Chapter 4.3.2 --- Alteration of the mRNA Expression of Calcium Binding ProteinS100B --- p.128 / Chapter 4.4 --- Alteration of Lipid Metabolism in Decreased Glucose Supply --- p.130 / Chapter 4.4.1 --- Up-regulation of ApoE mRNA Expression in Glucose Deficiency --- p.133 / Chapter 4.4.2 --- Cholesterol Homeostasis in Glial Cells --- p.133 / Chapter 4.4.3 --- Fatty Acids Homeostasis in Glial Cells --- p.135 / Chapter 4.4.4 --- Decreased Ketone Bodies synthesis in Glucose Deficiency --- p.143 / Chapter 4.5 --- Limitations of the Current Study --- p.144 / Chapter 4.6 --- Future Directions --- p.145 / Chapter Chapter 5 --- Conclusion --- p.147 / References --- p.148 / Appendix --- p.169