DEVELOPMENT, CHARACTERIZATION, AND IMPLEMENTATION OF AN IN VITRO MODEL OF CEREBROSPINAL FLUID OUTFLOW ACROSS THE ARACHNOID GRANULATIONS DISSERTATION Presented in Partial Fulfillment of the of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By David W. Holman, B.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Deborah Grzybowski, Advisor Dr. Rita Alevriadou ___________________________________ Dr. Mark Ruegsegger Advisor Biomedical Engineering Graduate Program ABSTRACT The arachnoid granulations (AGs) are herniations of the arachnoid membrane through the dura mater into the dural venous sinuses. They represent the final barrier between the cerebrospinal fluid (CSF) and venous blood and are involved in the return of CSF to the systemic venous circulation. Despite 125 years of study, our knowledge remains limited regarding the mechanism of fluid movement across the AGs. The location of the AGs within the cranial vault has made functional characterization of these structures difficult. In addition to their role in normal CSF turnover, the AGs may also play a role in disorders of CSF homeostasis. Of particular interest is idiopathic intracranial hypertension (IIH), where an increased intracranial pressure has been attributed to an increased resistance to CSF outflow, occurring at the AGs. The goal of this dissertation is to develop, characterize and implement an in vitro, model of CSF outflow across human AGs. This model can be utilized to study CSF outflow under normal and pathological conditions. This dissertation describes the culture and characterization of cells grown from human AG tissue. These AG cells expressed a unique immunological profile of cytoskeletal and junctional proteins that was ii maintained in culture. AG cells grown on permeable culture supports and perfused under pressures that mimic normal intracranial pressure showed a directionality of fluid transport that is consistent with the transport of CSF in vivo. Further, when fixed under pressure these cultured cells showed structures that are consistent with mechanisms of fluid transport described in intact AG tissue. Comparison of in vitro hydraulic conductivity values with known CSF parameters suggested that AG cells in culture can accurately represent the hydraulic conductivity of intact AG tissue in vivo. AG cells exposed to a physiological range of retinol concentrations, to replicate those seen in patients with primary IIH, showed no significant change in barrier integrity, indicating that retinol did not increase AG cell outflow resistance in culture. In summary, the results presented here demonstrate a working cell culture model of the CSF outflow pathway across the AGs that can be utilized to study CSF egress under normal and pathological conditions. iii Dedicated to my mother and father, Mike, Kathy, and Katie iv ACKNOWLEDGMENTS I would first like to thank my research advisor, Dr. Grzybowski, for her support and encouragement. I would also like to thank Drs. Alevriadou and Ruegsegger for graciously serving on my candidacy and dissertation committees and providing me with valuable feedback. I am also deeply indebted to Dr. Bhavya Mehta for mentoring and working patiently with me when I was starting this research project. I would like to give special thanks as well to Dr. Amy Long for her helpful discussions on vitamin A. I would like to acknowledge Dr. Lubow for his enthusiasm and unique perspective. I would like to thank Dr. Harrison for his valuable insight on working with vitamin A and for allowing me the generous use of his laboratory resources. Much of this research would not have been possible without help of numerous individuals. I would like to thank Jeff Pelley and the staff at the OSU Autopsy Center for their assistance in procuring tissue samples. I am also grateful to Kathy Wolken and Brian Kemmenoe of the Campus Microscopy and Imaging facility for their help and technical know-how in matters of imaging, often on short notice. I would like to acknowledge Melanie Senitko, Kirsten Gibbons, and David Morelli in the Biomedical Engineering Department for their help with scheduling, ordering, and deadlines. v I am grateful to have had the opportunity to work with many highly motivated students during my time at OSU. I would especially like to thank Bongsu Kim for his helpful discussions on research problems and his unique sense of humor. It was also a privilege to work with many great colleagues including Sean McClory, Shelley Glimcher, Matt Fleshman, Neal Mehan, and Joe Heacock. Finally, I would like to acknowledge and thank the Department of Ophthalmology and the Ohio Lions Eye Research Foundation for their generous support of my fellowship during my time at Ohio State. vi VITA May 30, 1980………………………………………………Born – Cincinnati, OH May 2003…………………………………………………..B.S. Chemical Engineering, The University of Virginia September 2003-present…………………………………..Graduate Research Fellow The Ohio State University PUBLICATIONS Li JZ, Holman DW, Li H, Liu AH, Beres B, Hankins GR, Helm GA. “Long-term tracing of adenoviral expression in rat and rabbit using luciferase imaging.” J Gene Med. June; 7(6): 792-802, 2005. Holman DW, Grzybowski DM, Mehta BC, Katz SE, Lubow M. “Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue.” Cerebrospinal Fluid Res 2:9, 2005. Grzybowski DM, Holman DW, Katz SE, Lubow M. “In Vitro Model of Cerebrospinal Fluid Outflow through Human Arachnoid Granulations” IOVS 47(8): 3664-3672, 2006. Mehta BC, Holman DW, Grzybowski DM, Chalmers JJ. “Characterization of arachnoidal cells cultured on three-dimensional non-woven PET matrix” Tissue Eng 13: 1269-79, 2007. FIELD OF STUDY Major Field: Biomedical Engineering Minor Field: Neural Hydrodynamics and Biological Transport vii TABLE OF CONTENTS ABSTRACT....................................................................................................................... ii ACKNOWLEDGMENTS .................................................................................................v VITA................................................................................................................................. vii LIST OF TABLES ...........................................................................................................xv LIST OF FIGURES ....................................................................................................... xvi CHAPTER 1 - INTRODUCTION....................................................................................1 1.1 Research Motivation................................................................................................1 1.2 Research Objective ..................................................................................................3 1.2.1 Culture and Characterization of Arachnoid Cells Grown from Human AG Tissue ...........................................................................................................................3 1.2.2 Development of an In Vitro System to Perfuse Cultured AG Cells Under Pressure........................................................................................................................4 1.2.3 Validation of In Vitro Model with In Vivo CSF Parameters.............................4 1.2.4 Implementation of In Vitro Model to Study Disorders of CSF Circulation .......4 1.3 Dissertation Organization .......................................................................................4 CHAPTER 2 - LITERATURE REVIEW........................................................................7 2.1 INTRODUCTION....................................................................................................7 2.2 GENERAL CEREBROSPINAL FLUID CIRCULATION.................................8 2.2.1 Composition of Cerebrospinal Fluid...................................................................8 2.2.2 Functions and Volume of CSF............................................................................8 2.2.3 Anatomy of the CSF System...............................................................................9 2.3 CSF FORMATION................................................................................................11 2.3.1 Choroid Plexus..................................................................................................11 2.3.2 Mechanism of CSF Secretion ...........................................................................12 2.3.3 Basolateral CPE Transporters and Channels ....................................................14 2.3.4 Apical CPE Transporters ..................................................................................14 viii 2.4 CSF ABSORPTION...............................................................................................15 2.4.1 Arachnoid Pathway of CSF Outflow ................................................................19 2.4.2 Driving Forces for CSF Outflow ......................................................................21 2.4.3 Outflow of CSF via Open or Closed Channels.................................................21 2.4.4 Ultrastructural Studies of Animal AG Tissue...................................................22 2.4.5 Mechanism of CSF Outflow via Giant Vacuoles .............................................24 2.4.6 Ultrastructural Studies of Human AG Tissue ...................................................26 2.4.7 Differences between Human