Loma Linda University TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works Loma Linda University Electronic Theses, Dissertations & Projects 3-2016 Role of Slit2 in Reducing Complications after Surgical Brain Injury Prativa Sherchan Follow this and additional works at: http://scholarsrepository.llu.edu/etd Part of the Medical Physiology Commons, Neurology Commons, and the Neurosciences Commons Recommended Citation Sherchan, Prativa, "Role of Slit2 in Reducing Complications after Surgical Brain Injury" (2016). Loma Linda University Electronic Theses, Dissertations & Projects. 329. http://scholarsrepository.llu.edu/etd/329 This Dissertation is brought to you for free and open access by TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. It has been accepted for inclusion in Loma Linda University Electronic Theses, Dissertations & Projects by an authorized administrator of TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. For more information, please contact [email protected]. LOMA LINDA UNIVERSITY School of Medicine in conjunction with the Faculty of Graduate Studies ____________________ Role of Slit2 in Reducing Complications after Surgical Brain Injury by Prativa Sherchan ____________________ A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Physiology ____________________ March 2016 © 2016 Prativa Sherchan All Rights Reserved Each person whose signature appears below certifies that this dissertation in his/her opinion is adequate, in scope and quality, as a dissertation for the degree Doctor of Philosophy. , Chairperson John H. Zhang, Professor of Anesthesiology, Neurosurgery, Neurology and Physiology Daila Gridley, Professor of Radiation Medicine and Basic Sciences Richard Hartman, Professor of Psychology Andre Obenaus, Professor of Pediatrics, Associate Research Professor of Behavioral Health and Associate Professor of Basic Sciences Jiping Tang, Professor of Basic Sciences iii ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to my mentor Dr. John H. Zhang for his support, patience and guidance throughout my training. You have always inspired me to drive myself to explore the depths of scientific research. I am deeply grateful for the numerous opportunities that you have provided me with to advance not only my scientific knowledge and skills but to gain valuable leadership and managerial skills. Thank you, Dr. Zhang for your continued support. I would also like to thank my committee members Dr. Jiping Tang, Dr. Daila Gridley, Dr. Richard Hartman, and Dr. Andre Obenaus for their time, valuable advice and support. To members of Zhang Neuroscience Laboratories past and present, I would like to thank you for your friendship and the opportunities to collaborate. I am thankful for friends that I have made and the memorable times that we have spent together. To my family and friends, your love and support gives me the strength to stand up to any challenge that comes my way. I could not have done this without you. I want to thank my parents for the sacrifices that they have made so that I could have a great education and all the opportunities for success. My sister and brother, you are the rock in my life. Thank you for always being there. My little son Sid, you are the joy of my life and you make everything worth trying for. Thank you for bringing smiles to my life every day. iv CONTENT Approval Page ...................................................................................................... iii Acknowledgements .............................................................................................. iv List of Figures ....................................................................................................... ix List of Tables ........................................................................................................ x List of Abbreviations ............................................................................................. xi Abstract .............................................................................................................. xiii Chapter 1. Introduction to Surgical Brain Injury ........................................................... 1 Surgical Brain Injury ............................................................................. 2 Pathophysiology of Surgical Brain Injury .............................................. 3 Brain Edema ................................................................................... 3 Neuroinflammation .......................................................................... 3 Surgical Brain Injury Rat Model ............................................................ 4 Experimental Therapeutics for Surgical Brain Injury ............................. 8 Vascular Endothelial Growth Factors .............................................. 8 Matrix Metalloproteinases ............................................................... 9 Cyclooxygenase-2 ......................................................................... 11 Conclusion ......................................................................................... 12 References ......................................................................................... 14 2. Role of Slit2 in CNS Pathology ................................................................ 18 Introduction to Slit2-Robo ................................................................... 18 Role of Slit2 During CNS Development .............................................. 19 Role of Slit2 in CNS Disorders ........................................................... 20 Glioma ........................................................................................... 20 Traumatic and Surgical Brain Injury .............................................. 23 Cerebral Ischemia ......................................................................... 24 Intracerebral Hemorrhage ............................................................. 24 Epilepsy ........................................................................................ 25 v Role of Slit2 Dependent of Cell Types ............................................... 26 Endothelial Cells ........................................................................... 26 Pericytes ....................................................................................... 28 Regulation of Slit2-Robo in the CNS .................................................. 29 Transcription Factor Pax6 ............................................................. 29 MicroRNA 218 ............................................................................... 30 Endocannabinoids and Cannabinoid Receptors ........................... 31 Downstream Targets of Slit2-Robo Signaling in the CNS ................... 31 Small Rho-GTPase Cdc42 ............................................................ 31 Focal Adhesion Kinase ................................................................. 32 Src and Extracellular Signal-Regulated Kinase 1/2 ....................... 33 Research Question, Hypothesis and Research Aims ......................... 33 References ......................................................................................... 37 3. Recombinant Slit2 Attenuates Neuroinflammation After Surgical Brain Injury by Inhibiting Peripheral Immune Cell Infiltration via Robo1-srGAP1 Pathway in a Rat Model ................................................. 41 Abstract .............................................................................................. 42 Introduction ......................................................................................... 44 Materials and Methods ....................................................................... 46 Animals ......................................................................................... 46 Experimental Design ..................................................................... 46 Surgical Brain Injury Model ........................................................... 48 Intracerebroventricular Injection .................................................... 48 Brain Water Content Measurement ............................................... 49 Assessment of Neurological Function ........................................... 49 Enzyme Linked Immunosorbent Assay ......................................... 50 Western Blotting ............................................................................ 50 Immunohistochemistry .................................................................. 51 Cdc42 Activity Assay ..................................................................... 52 Statistical Analysis ........................................................................ 52 Results .............................................................................................. 53 Temporal Expression and Localization of Slit2 After SBI .............. 53 Temporal Expression and Localization of Robo1 After SBI ........... 55 vi Endogenous Slit2 Knockdown Worsened Outcomes After SBI ................................................................................................ 57 Recombinant Slit2 Administration Reduced Brain Edema and Improved Neurological Function 24 and 72 hours after SBI ................................................................................................ 59 Recombinant Slit2 Reduced Neuroinflammation