Brain 3-Mercaptopyruvate Sulfurtransferase (3MST): Cellular Localization and Downregulation after Acute Stroke Zhao Heng (B Sc (Hons.), Fudan University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Zhao Heng 3rd July, 2013 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my supervisor, Professor Wong Tsun Hon, Peter He has been a fantastic mentor who provided me with the opportunity to continue my graduate studies and guided me throughout the whole project I am greatly inspired by his dedication to academic and research works He has always been extraordinarily excellent at managing both academic and research tasks, which has in turn motivated me in efficiently handling mine In addition, I would like to thank Professor Wong for supporting me in several scientific conferences and training programs My appreciation is also extended to my co-supervisor, Associate Professor Ng Yee Kong, for his scientific advice and continuous support over all these years This thesis could not have been written without his valuable insights and suggestions I would like to extend my gratitude to all the members of the lab, past and present, for their help and support throughout these years I am especially grateful to Mrs Ting Wee Lee, our outstanding laboratory technologist, for her assistance on filing paper works, ordering chemicals and managing plenty of administrative stuffs Also, I would like to express my sincere appreciation to Miss Chan Su Jing As a mentor, her invaluable experience in conducting experiments has tremendously facilitated the whole process of my study Additionally many thanks to Professor Eng H Lo and Associate Professor Ming Xian for their support and guidance on the collaborations in the United States Sincere appreciation to Dr Mie Yamamoto, Dr Emiri Mandeville and Dr Elga Esposito for their technical helps Heartfelt gratitude to Miss Wu Qi, Miss Yang Ying, Miss Koh Shu Qing and Miss Lim Tze Wei for the support and friendships over the years Finally, I wish to thank my parents, my wife and the other family members for their support and encouragement throughout these years TABLE OF CONTENTS SUMMARY VI LIST OF TABLES VIII LIST OF FIGURES IX LIST OF SYMBOLS XII PUBLICATIONS XVI CHAPTER 1: Brain 3-Mercaptopyruvate Sulfurtransferase (3MST): Cellular Localization and Downregulation after Acute Stroke 1 INTRODUCTION 1.1 Stroke 1.1.1 Epidemiology 1.1.2 Classification 1.1.3 Risk Factors 1.1.4 Prognosis 1.1.5 Therapy 1.1.5.1 Ischemic Stroke 1.1.5.1.1 Thrombolysis 1.1.5.1.2 Aspirin 1.1.5.1.3 Thrombectomy 1.1.5.2 Hemorrhagic Stroke 1.1.6 Prevention 1.1.6.1 Primary Prevention 1.1.6.2 Secondary Prevention 1.1.7 Experimental Models 1.1.7.1 In Vivo Models 1.1.7.1.1 Global 10 1.1.7.1.2 Focal 11 1.1.7.1.2.1 Permanent Transcranial Model 12 1.1.7.1.2.2 Filament Model 12 1.1.7.1.2.3 Thromboembolic Model 13 1.1.7.2 In Vitro Models 14 1.1.7.2.1 Oxygen Glucose Deprivation 15 I 1.2 Hyperhomocysteinemia 16 1.2.1 Risks 18 1.2.1.1 Cardiovascular Disease 18 1.2.1.1.1 Pathophysiologic Mechanism 18 1.2.1.1.2 Epidemiological Studies 20 1.2.1.2 Neurodegenerative Disease 21 1.2.1.2.1 Alzheimer’s Disease 21 1.2.1.2.2 Stroke 22 1.2.2 Causes 23 1.2.3 Therapy 25 1.2.4 Metabolism 28 1.2.4.1 Homocysteine 28 1.2.4.2 Cysteine 29 1.2.4.2.1 Pyridoxal-5′-Phosphate (PLP)-Dependent Enzymes 30 1.2.4.2.1.1 Cystathionine β-Synthase 30 1.2.4.2.1.2 Cystathionine γ-Lyase 32 1.2.4.2.2 3-Mercaptopyruvate Sulfurtransferase 32 1.3 Hydrogen Sulfide 34 1.3.1 Properties 34 1.3.2 Toxicity 35 1.3.3 Endogenous H2S 36 1.3.4 Physiological Role 38 1.3.4.1 Nervous System 39 1.3.4.1.1 Neuromodulator 39 1.3.4.1.2 Neuroprotectant 41 1.3.4.2 Smooth Muscle 44 1.3.5 1.4 Biosynthesis 44 Knowledge Gaps and Study Objectives 45 MATERIALS AND METHODS 48 2.1 Animals 48 2.2 Middle Cerebral Artery Occlusion 48 2.2.1 Permanent Middle Cerebral Artery Occlusion Model 48 2.2.2 Filament Middle Cerebral Artery Occlusion Model 49 2.2.3 Thromboembolic Middle Cerebral Artery Occlusion Model 51 II 2.2.3.1 Preparation for Thromboembolic Model 51 2.2.3.1.1 Homologous Clot Preparation 51 2.2.3.1.2 PE-50 Catheter Modification 51 2.2.3.2 Thromboembolic Model 52 2.2.3.2.1 Settlement and Measurement of rCBF 52 2.2.3.2.2 Physiological Monitoring and Venous Line for Injection 52 2.2.3.2.3 Preparation for Injecting the Clot 52 2.2.3.2.4 Thromboembolic Model 55 2.2.4 Measurement of Infarct Volumes 57 2.2.5 Neurological Evaluation 59 2.3 Brain perfusion 62 2.3.1 Prepare Apparatus and Anesthesia 62 2.3.2 Perfusion Surgery 62 2.3.3 Perfusion 63 2.4 Brain Dissection 64 2.4.1 Brain Removal 64 2.4.2 Dissection 64 2.5 Primary Astrocyte Culture 65 2.5.1 Brain Dissection 65 2.5.2 Tissue Digestion 66 2.5.3 Culture purification 66 2.6 Oxygen-Glucose Deprivation 67 2.7 Immunohistochemistry 67 2.7.1 Fluorescent Staining 67 2.7.2 DAB Staining 68 2.8 Western Blot Analysis 69 2.9 H2S-Producing Enzyme Assay 70 2.9.1 3MST Assay 70 2.9.2 CBS Assay 71 2.10 Fluorescent Staining of H2S 71 2.10.1 Synthesis of Probe 72 2.10.2 Reaction of H2S Probe with H2S 73 2.10.3 Experimental Procedure 74 2.10.3.1 Determination of Wavelength for H2S Detection 74 III 2.10.3.2 H2S Standard Curve 74 2.10.3.3 H2S in the Cells 75 2.11 Statistical Analysis 75 RESULTS 76 3.1 H2S-Producing Enzyme Activities 76 3.1.1 H2S Concentration Standard Curve 76 3.1.2 3MST Activities in the Brain 78 3.1.3 CBS Activities in the Brain 80 3.2 3MST Regional and Cellular Localization 83 3.2.1 3.3 3MST Expression in Mice 88 3MST Expression after pMCAO 92 3.3.1 3MST Expression in Cortex 93 3.3.1.1 3MST Expression at Various Time Points in Cortex 93 3.3.1.2 3MST Expression at 24h after pMCAO in Cortex 96 3.3.1.3 3MST Expression at 72h after pMCAO in Cortex 98 3.3.2 3MST Expression in Striatum 99 3.3.2.1 3MST Expression at Various Time Points in Striatum 99 3.3.2.2 3MST Expression at 24h after pMCAO in Striatum 101 3.3.2.3 3MST Expression at 72h after pMCAO in Striatum 102 3.3.3 3MST Expression in Corpus Callosum 103 3.3.3.1 3MST Expression at 24h after pMCAO in Corpus Callosum 103 3.3.3.2 3MST Expression at 72h after pMCAO in Corpus Callosum 104 3.4 3MST Expression in Cerebellum and Midbrain 105 3.5 3MST Expression in Primary Astrocytes under OGD 108 3.6 H2S Concentration Determination 113 3.6.1 Determination of Wavelength for H2S Detection 113 3.6.2 H2S Standard Curve 115 3.6.3 H2S Concentration Determination in Primary Astrocytes 117 DISCUSSION 119 4.1 Ischemic Model Selection 119 4.2 H2S-Producing Enzymes 120 4.3 3MST Localization 122 4.4 3MST and CBS Activity 124 4.5 Roles of Astrocytes 125 IV 4.6 H2S Probe 126 4.7 Limitations 129 CHAPTER 2: Vasculome Mapping for Biomarkers in Mild Traumatic Brain Injury 132 INTRODUCTION 132 1.1 Epidemiology 132 1.2 Classification 133 1.3 Risk Factors 134 1.4 Pathophysiology 134 MATERIALS AND METHODS 135 2.1 Animals 135 2.2 Closed Head Injury Model 136 2.3 Behavioral Tests 137 2.3.1 Neurological Severity Score 137 2.3.2 Wire Grip Test 139 2.3.3 Corner Test 140 2.3.4 Foot Fault Test 140 2.3.5 Y-Maze Test 140 RESULTS 142 3.1 Motor Assessment 142 3.2 Cognitive Assessment 148 DISCUSSION 150 REFERENCES 152 V SUMMARY Background and Purpose It has been reported that poor clinical outcome in acute stroke patients is strongly associated with high plasma homocysteine and cysteine levels The administration of cysteine increased the infarct volume after experimental stroke induced by pMCAO As CBS can produce H2S using Cys and/or Hcy as substrates, these observations indicate that the Cys effect may be due to its conversion to H2S Moreover, administration of NaHS, a donor of H2S, increased infarct volume after pMCAO It is known that H2S may be produced by CBS and 3MST along with CAT However, it is not known what changes occur in 3MST expression under ischemic conditions in the brain As H2S is known to increase after stroke, we hypothesized that the expression of 3MST might increase if 3MST is the major source of H2S under such conditions Materials and Methods The regional distribution of 3MST activities and the cellular localization of 3MST were measured in normal rat brains And the expressions of 3MST in the cortex, striatum and corpus callosum were investigated before and after pMCAO In vitro studies were also done in primary astrocytes including OGD and H2S concentration determination Results No significant differential distribution of 3MST activities in all regions exhibiting mean activities in the range of about 21 to 26 µmol/g tissue/h and 3MST activities were almost completely inhibited in the presence of 2ketobutyric acid And 3MST was 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Cortex 93 3 .3. 1.2 3MST Expression at 24h after pMCAO in Cortex 96 3. 3.1 .3 3MST Expression at 72h after pMCAO in Cortex 98 3. 3.2 3MST Expression in Striatum 99 3. 3.2.1 3MST Expression