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Gene Expression Profile in the Middle Cerebral Artery View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarBank@NUS GENE EXPRESSION PROFILE IN THE MIDDLE CEREBRAL ARTERY AND FRONTAL CORTEX OF HYPERTENSIVE RABBITS JIN SHALAI (B.Sc. (Biological Sciences), Zhejiang University, China) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY 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. Jin Shalai 10.3.2014 1 ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to my supervisor, Associate Professor Ong Wei Yi, Department of Anatomy, National University of Singapore, for suggesting this study topic and his patient guidance and encouragement throughout the study. During my postgraduate study at National University of Singapore, his invaluable supervisions and enlightening ideas has not only introduced me to a new research field but has also showed me how to be a real scientific researcher. I am grateful to my seniors, Dr Kazuhiro Tanaka, Kim Ji Hyun, Ma May Thu, Poh Kay Wee, and Mary Pei-Ern Ng, for their encouragement, technical help and critical comments. Sincere appreciation also to all the laboratory members in Histology Lab, Neurobiology Programme, Centre for Life Science, National University of Singapore: Tan Yan, Yang Hui, Chew Wee Siong, Ee Sze Min, Loke Sau Yeen, Yap Mei Yi Alicia; for their technical support, assistance in various aspects as well as their warm friendship. Their presence has made the laboratory an enjoyable place to work in. The accomplishment of this thesis could not exist without their help. Finally but not the least, I would also like to take this opportunity to thank my family members and friends for their constant support and encouragement. 1 TABLE OF CONTENTS ACKNOWLEDGEMENTS................................................................................ 1 TABLE OF CONTENTS ....................................................................................2 SUMMARY......................................................................................................... 4 LIST OF FIGURES.............................................................................................5 LIST OF TABLES.............................................................................................. 6 ABBREVIATIONS............................................................................................. 7 CHAPTER 1 INTRODUCTION....................................................................... 9 1.1 Stroke............................................................................................................ 10 1.2 Hypertension................................................................................................. 11 1.3 Middle Cerebral Artery................................................................................. 13 1.4 Frontal Cortex............................................................................................... 15 1.5 Animal Model of Hypertension.................................................................... 16 1.6 Aim of the Study........................................................................................... 21 CHAPTER 2 MATERIALS AND METHODS…………..…...…………….22 2.1 Rabbits and treatment.................................................................................... 23 2.2 Serum Cholesterol......................................................................................... 24 2.3 RNA extraction............................................................................................. 25 2.4 cDNA Synthesis............................................................................................ 27 2.5 Microarray Analysis...................................................................................... 27 2 2.6 Pathway and network.................................................................................... 29 2.7 Real-time PCR analyses.................................................................................. 29 2.8 Western Blot.................................................................................................. 34 CHAPTER 3 RESULTS…………………………........................................... 37 3.1 Body Weight................................................................................................. 38 3.2 Serum Cholesterol and Mean Arterial Pressure............................................ 39 3.3 Microarray data collection and analysis........................................................ 41 3.3.1 Differentially expressed genes found in MCA……………...………...42 3.3.2 Differentially expressed genes found in FC………..…………………46 3.3.3 Differentially expressed genes found in common…….……..………..51 3.4 Pathway and network analyses...................................................................... 56 3.5 Real-Time PCR............................................................................................. 60 3.6 Western Blot.................................................................................................. 61 CHAPTER 4 DISCUSSION AND CONCLUSION...................................... 66 4.1 Discussion .................................................................................................... 67 4.2 Limitation and future study........................................................................... 73 4.3 Conclusion………………….………..…………………….….…………….74 REFERENCES.................................................................................................. 75 3 SUMMARY Hypertension is known to contribute to the progression of plaque formation in hyperlipidemia and is an important risk factor in cerebral atherosclerosis and stroke. This study presents an expression profile of various genes that are involved and regulated in hypertension in both intra- and extracranial vessels. The expression of genes and extension of damage to cerebral arteries caused by hypertension were evaluated quantitatively and morphologically in 10 New Zealand White rabbits with and without hypertension, induced using the 2- kidney, 1-clip Goldblatt hypertension model. Genes in the frontal cortex and middle cerebral artery respectively were filtered from microarray analysis and subjected to the Ingenuity Pathway Analysis where canonical pathways and a network of other genes related to our gene input were generated. From the selection of our genes of interest, 8 genes PPARA, PRL-R, PTGDR, P450, Gab3, Tnfs14, Sell and Lass3 were verified by RT-PCR.These genes have shown to be involved in the progression or contribution towards inflammatory diseases such as atherosclerosis. PPARA is a major regulator of lipid metabolism. Gab3 and Tnfs14 produce cytokines and chemokines while P450 protein is known to increase metabolism of arachidonic acid, a precusor in the production of eicosanoids which involves PTGDR. In addition, Tnfs14 and Sell are involved in endothelial cell adhesion, activation and disruption. Endothelial dysfunction is a hallmark for vascular diseases, and is often regarded as a key early event in the 4 development of atherosclerosis thus further study of these genes may lead to a better understanding on the role of hypertension in stroke. List of Figures Figure A: Outer surface of cerebral hemisphere................................................. 14 Figure B: The arterial circle and arteries of the brain......................................... 14 Figure C: Quick concept for the two kidneys, one clip model............................ 18 Figure 1A: Weight chart of rabbits …………………........................................ 38 Figure 1b: Serum cholesterol levels in rabbits ………………………..………..39 Figure 1c: Mean arterial pressure (MAP) levels in rabbits………..................... 40 Figure 2: Venn diagram summarizing genes ………......................................... 42 Figure 3: Network of genes mapped in middle cerebral artery…………......…..57 Figure 4: Network of genes mapped in frontal cortex ………........................... 58 Figure 5: Network of common genes ………..................................................... 59 Figure 6: Real-Time PCR results ………........................................................... 60 Figure 7: Western blot analysis………………………………..…..………..…..62 Figure 8: Calculation of the gene expression ………......................................... 63 5 List of Tables Table A: Pathophysiology of renovascular hypertension................................... 19 Table B: gene selection for RT-PCR...................................................................33 Table C: Concentration of primary and secondary antibodies............................ 36 Table 1: Up regulated genes in the middle cerebral artery................................. 43 Table 2: Down regulated genes in the middle cerebral artery.............................45 Table 3: Up regulated genes in the frontal cortex............................................... 48 Table 4: Down regulated genes in the frontal cortex.......................................... 50 Table 5: Up regulated genes common in the middle cerebral artery and frontal cortex................................................................................................................... 52 Table 6: Down regulated genes common in the middle cerebral
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