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Characterisation of Inflammatory Responses in Two Models of Experimental Ischaemia

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Characterisation of Inflammatory Responses in Two Models of Experimental Ischaemia CHARACTERISATION OF INFLAMMATORY RESPONSES IN TWO MODELS OF EXPERIMENTAL ISCHAEMIA Louise Marks, April 2001 A thesis submitted for the degree of Doctor of Philosophy to the Faculty of Medicine, University of Glasgow. Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Bearsden Road, Glasgow, G61 1QH. UNIVERSITY GLASGOW ProQuest Number: 13833994 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13833994 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 \lbl2 0)P1\ II Contents Title page page I Contents page n List of tables page VIII List of figures page IX Abbreviations page XIV Acknowledgements page XVI Authors declaration page XVH Summary page XVIII Chapter 1. Introduction page 1 1.1 Stroke Background page 1 1.1.1 Stroke facts and figures page 1 1.1.2 Ischaemia and stroke page 1 1.2 Classification of stroke page 2 1.2.1 Haemorrhagic stroke page 3 1.2.2 Ischaemic stroke page 3 1.3 Models of cerebral ischaemia page 4 1.3.1 Use of the rat as a model of cerebral ischaemia page 4 1.3.2 Rat models of experimental ischaemia page 7 1.3.3 Models of permanent middle cerebral artery occlusion page 9 1.3.4 Models of transient focal ischaemia page 12 Ill 1.3.5 Models of global ischaemia page 16 1.3.6 The spontaneously hypertensive stroke prone rat page 18 as a model of cerebrovascular disease 1.4 Ischaemic events page 19 1.4.1 Reperfusion injury page 21 1.4.2 Excitotoxicity and ischaemia page 22 1.4.3 Inflammation and ischaemia page 23 1.4.3.1 Neutrophils page 26 1.4.3.2 Microglia page 31 1.4.3.3 Matrix metalloproteinases page 48 1.4.3.4 Free radicals page 72 1.4.3.5 Calcium page 79 Chapter 2 Materials and methods page 81 2.1 Surgical procedures page 81 2.1.1 Preparation of animals for surgery page 81 2.1.2 Intraluminal thread model of ischaemia page 82 2.1.3 Permanent ischaemia by electrocoagulation page 85 2.1.4 LPS stereotaxic injections page 86 2.1.5 Recovery of animals following surgery page 87 2 .2 Processing of tissue for H&E and immunohistochemistry page 88 2.2.1 Fixation page 88 2.2.2 Post fixation page 90 2.2.3 Cutting cryostat sections page 90 2.2.4 Paraffin processing page 91 2.2.5 Infarct determination page 91 2.2.5.1 Haematoxylin & eosin page 91 2.2.5.2 Detection of ischaemic neurons page 92 2.2.5.3 Measuring of infarct size page 94 2.2.6 Immunohistochemistry page 96 2.2.6.1 On cryostat cut sections page 96 2.2.6.2 Dehydration and counterstaining page 98 2.2.6.3 On paraffin sections page 98 2.2.6.4 Double label immunohistochemistry page 99 2.2.6.5 Fluorescence labelling page 101 2.2.7 Controls for immunohistochemistry page 102 2.3 Western blotting page 102 2.3.1 Mini gels page 102 2.3.2 Protein determination page 103 2.3.3 Running of gels page 104 2.3.4 Optical density measurements page 106 2.4 Scanning Electron Microscopy page 106 2.4.1 Fixation page 106 2.4.2 Processing of tissue page 107 2.4.3 Developing of films page 109 V Results : Chapter 3. Microvilli/ Neutrophil Study page 110 3.1 Introduction page 110 3.2 Materials and Methods page 112 3.2.1 Surgical procedures page 112 3.2.2 Neutrophil adherence/ accumulation page 113 3.2.3 Ultrastructural changes page 113 3.2.4 Statistical analysis page 114 3.3 Results page 114 3.3.1 Physiological variables page 114 3.3.2 Extent of ischaemic damage page 116 3.3.3 Neutrophil adhesion page 117 3.3.4 Microvilli counts page 118 3.3.5 Ultrastructural changes to MCA page 124 3.4 Discussion page 125 Results: Chapter 4. Matrix Metalloproteinase Study page 136 4.1 Introduction page 136 4.2 Materials and methods page 138 4.2.1 Surgical procedures page 138 4.2.2 Infarct determination page 139 4.2.3 Western blotting page 139 4.2.4 Immunohistochemistry page 139 4.2.5 Double label immunohistochemistry page 140 4.2.6 Blocking peptide control page 141 VI 4.2.7 Distribution and quantification of staining page 141 4.2.8 Statistical analysis page 142 4.3 Results page 143 4.3.1 Physiological variables page 143 4.3.2 Infarct size page 143 4.3.3 Western blotting- mini gels page 145 4.3.4 Distribution and cellular location of MMP-9 page 149 4.3.5 Distribution and cellular location of MMP-8 page 156 4.3.6 Specificity of neuronal staining page 159 4.3 .7 BBB permeability and MMP staining page 161 4.4 Discussion page 163 Results: Chapter 5. SHRSP / WKY Microglial Study page 173 5.1 Introduction page 173 5.2 Materials and methods page 176 5.2.1 Surgical procedures page 177 5.2.2 Infarct determination page 177 5.2.3 Neutrophil counts page 178 5.2.4 Immunohistochemistry page 179 5.2.5 Quantification of staining page 179 5.2.6 Statistical analysis page 181 5.3 Results page 182 5.3.1 Physiological parameters page 182 5.3.2 Infarct size page 182 VII 5.3.3 Neutrophil counts page 186 5.3.4 Characterisation of microglial activation page 186 5.3.5 Distribution of microglia page 187 5.3.6 Microglial counts page 189 5.3.7 Strain differences page 189 5.3.8 Correlation graphs page 197 5.3.8 naive controls page 197 5.4 Discussion page 201 Results: Chapter 6 SHRSP/WKY Time Course Study page 208 6.1 Introduction page 208 6.2 Materials and methods page 209 6.2.1 Assessment of brain swelling page 209 6.3 Results page 210 6.3.1 Physiological variables page 210 6.3.2 Evolution of infarct over 72-hour time course page 210 6.3.3 Tissue Swelling page 213 6.3.4 Microglial counts in brain regions page 215 6.3.5 Characterisation of microglial response page 225 6.3.6 Microglial distribution maps page 228 6.3.7 Characterisation of MMP-8 /MMP-9 staining page 232 6.3.8 Distribution maps for MMP staining page 239 6.3.9 Characterisation of IL-1 p staining page 244 6.4 Discussion page 246 VIII Chapter 7 General Discussion page 257 7.1 Introduction page 257 7.2 Microvilli and neutrophils in ischaemic damage page 258 7.3 Microglia in inflammatory mediated ischaemic damage page 261 7.4 MMP-8 and MMP-9 in ischaemic damage page 264 7.5 General conclusions page 265 References page 267 Publications page 299 Appendices pagel-VII IX List of tables 1. Role of cytokines in the proliferation of microglia page 38 2. Involvement of microglia in other pathological conditions page 46 3. Antibodies against microglia page 47 4. The matrix metalloproteinases family page 51 5. MMP localisation within cell types page 58 6. MMP studies using various animal models of experimental ischaemia page 63 7. Physiological variables for scanning electron microscopy page 114 8. Number of microvilli per 25 pm2 in ILT induced ischaemia page 121 9. Physiological variables for 24 hour ILT induced ischaemia page 144 10.Physiological variables for 24 hour permanent MCAO page 183 11 .Physiological variables for time course study of permanent MCAO page 211 X List of Figures 1. Arrangement of the blood vessels in the Circle of Willis page 5 2. Infarction rate is determined by the site and location of the area of electrocoagulation page 10 3 . Intraluminal thread induced ischaemia page 13 4. Events following ischaemia page 20 5. Inflammatory time scale following ischaemia page 25 6. Steps of neutrophil migration page 27 7. Stages of microglial activation page 34 8. The migration of cells page 39 9. Structure of the extracellular matrix page 50 10. MMP structure page 53 11. Activation of MMPs page 56 12. Free radical involvement in ischaemia and reperfusion page 74 13 .a. Haber Weiss reaction, b. iron catalyses OH production, c. Fenton reaction. page 75 14. Involvement of calcium in reperfusion injury page 80 15. Intraluminal thread model of ischaemia page 83 16. Permanent middle cerebral artery occlusion by electrocoagulation page 85 17. Haematoxylin and eosin staining page 92 18. Morphology of ischaemic neurons page 93 19. Line diagram representing the eight coronal levels page 95 20. Processing of tissue for Scanning Electron Microscopy page 108 21. Microvilli in a control non-ischaemic vessel in the cortex page 111 22. Volume of ischaemic damage for intraluminal thread induced ischaemia. page 116 23. Lack of neutrophil accumulation in parenchymal vessels or MCAs page 117 24. Microvilli in parenchymal cortical vessels page 118 25. Number of microvilli per 25 pm2 in cortical parenchymal blood vessels page 119 26. Number of microvilli in small and large vessels page 120 27.
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