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Bell & Howell Information and Learning 300 North Zeeb Road, Ann Arbor, MI 48106-1346 USA 800-521-0600 STROKEIN-A-DISH: AN IIY WTRO MODEL OF MICROGLIAL ACTWATION AND STROKE Lipi Roy A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto @ Copyright by Lipi Roy 1998 National Library Biblioth&que nationale 191 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON KIA ON4 Ottawa ON K1 A ON4 Canada Canada The author has granted a non- L'autem a accorde me licence non exclusive licence dowing the exclusive pennettant a la National Library of Canada to Bibliotheque nationale du Canada de reproduce, loan, distribute or sell reproduire, peter, distribuer ou copies of this thesis in microform, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/^ de reproduction sur papier ou sur format electronique. The author retains ownership of the L'auteur conserve la propriete du copyright in this thesis. Neither the droit d'auteur qui protege cette these. thesis nor substantial extracts fiom it Ni la these ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent &e Imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Title: Stroke-In-A-Dish: An In Vipo Model of Microglial Activation and Stroke Name: Lipi Roy Department: Physiology Degree: Master of Science University of Toronto 1998 Microglia, immune cells of the brain, are implicated in several central nervous system disorders, including stroke. We have developed a simplified in vim model to study specific aspects of microglial activation: respiratory burst (reactive oxygen intermediate production) and morphology. A microscope-based imaging system was used to monitor morphology and fluorescence intensity before, during and after ischemic injury in cultured rat microglia. Long-term ischemia (15 hr) led to microglia death, whereas short-term ischemia (1 5 min) was a practical experimental paradigm that simulated an acute ischemic injury. The phorbol ester, PMA, increased respiratory burst by 56% and was, thus, used as a positive control to validate my model. PMA also induced morphological changes in microglia. Respiratory burst remained constant with the inactive analog, 4dM.A. Ischemia induced morphological changes but did not cause a respiratory burst. Two K+-channel inhibitors decreased respiratory burst. Viability tests revealed that microglia were alive following all treatments. The successll completion of a graduate degree is an accomplishment that cannot take place without the involvement of several individuals. The completion of my own Master's thesis is no exception. Within the lab environment, proper research and experimentation could not have taken place without the invaluable technical support provided by Lime Chen, Brent Clark and George Sakellaropoulos. The smooth operation of the computer network, electronics and hardware equipment could not have been possible without Brian Brown, Adam Charlinski and Frank Vidic. The dedicated efforts of the administrative staff of Playfair (Lyssa Cheng, Karen MacDonald and Rita Seto) and Physiology (Helena Hallett and Julie Weedmark) did not go unnoticed and were greatly appreciated. I also wish to acknowledge my advisory committee, Drs. Owen Jones and Linda Mills, and the graduate co-ordinator, Dr. Umberto Deboni for guiding me throughout my thesis; and Drs. Peter Pemefather and Frances Skinner for their data analysis wisdom. A big thank-you to my fellow graduate students - Francisco Cayabyab, Martin Chang, Rajesh Khanna, Bob Kotecha and Tim Mertens - for making many memorable moments in the lab. I owe a special gesture of appreciation to my supervisor, Dr. Lyanne Schlichter, who took me in as a graduate student, introduced me to the vast field of neurophysiology, enhanced my scientific writing skills and revised the content and organization of my thesis above and beyond the expectations of a graduate supervisor. Last, but never least, is the priceless presence of my family. The enduring care and wisdom of my mother and father enabled me to pursue my research endeavours. Though never in the lab, their financial and emotional support and prayers provided me with the strength, kindness and respect needed to succeed - not only in graduate school but an entire lifetime. Thank you. .. Abstract .............................................................................................................................~....u Acknowledgements...... ... ............................................................................................. IU Table of Contents................................................................................................................. iv .. List of Figures .................................................................... ..............................................vu List of Tables ................... ..... ...................................................................................ix List of Abbrevrahons. ...... .. ............................................................................................... x 1. What are Microglia? ......................................................................................................1 2 . Microgiial Morphology vs .Activation Status .............................................................. -2 3 . Stroke and Ischemia....................................................................................................... -4 i . In Vivo Evidence of Microglia Involvement in Stroke.................................... 4 ii. In Viho Evidence of Microglia Involvement in Stroke.................................... 4 iii . Microglia-Derived Cytotoxins ......................................................................... -5 iv . Microglia-Derived Cytokines and Growth Factors ............................................. 6 4. A Chemical Stimulus ..................................................................................................... 10 Rationale and Pathway......................................................................................... 10 5 . Ionchannels .................................................................................................................. 11 i . Physiology of Ion Channels .................... ..... ....................................................11 ii . Significance of Ion Channels in Respiratory Burst ........................................... 12 6 . Respiratory Burst andNADPH Oxidase..................................................................... 13 . 7. Obj ectwes and Hypotheses............................................................................................ 17 1 . CeUCulture ...........................................................-....................................................... 18 i . Isolation .......................................................................................................... 18 ii... Endotoxin-Free Equipment and Media ............................................................. 9 111. Cell Identification ............................................................................................ 20 2 . Respiratory Burst Dyes ................................................................................................. 24 i . Dye Selectionand Working Conditions ........................................................... 24 ii . Technical Difficulties and Solutions................................................................ -24 iii .Fluorescence Acquisition and Analysis ........................................................... -25 3. A Chemical Stimulus .................................................................................................... 25 4 . Developing the "Stroke-in-a-Dish" Model ................................................................... 26 5. Long-term Ischemia ................................................................................................... 28 7 . Chemicals