Differential Loss of Bidirectional Axonal Transport with Structural Persistence Within The Same Optic Projection of the DBA/2J Glaucomatous Mouse A Thesis Presented in partial fulfillment of the requirements for the degree of Masters of Science in the College of Graduate Studies of Northeast Ohio Medical University Matthew A. Smith B.A. Integrated Pharmaceutical Medicine Northeast Ohio Medical University 2014 Thesis Committee: Dr. Samuel D. Crish (advisor) Dr. Christine Crish Dr. Denise Inman Copyright Matthew A. Smith 2014 Abstract Glaucoma, the second leading cause of blindness worldwide, involves the degeneration of retinal ganglion cell bodies and their axons resulting in progressive vision loss. Much like other neurodegenerations, deficits in axonal transport are an early manifestation in the pathological progression of glaucoma. Previous studies suggest that anterograde and retrograde transport are differentially challenged and pre-degenerative in glaucoma, yet, both forms of transport have never been assessed within the same animal. We used a modified surgical procedure to assess retrograde transport while preserving the structure of the superior colliculus (SC) for both anterograde transport and immunohistochemical analysis in the same optic projection. Our findings demonstrate a 3-fold greater reduction in anterograde transport compared to retrograde transport in 9-10 month old animals. Retrograde transport remained largely intact until 13 months of age, where a reduction similar to anterograde transport was observed. Additionally, immunohistochemical staining revealed that retinal ganglion cell (RGC) axons remained intact until 13 months despite these early transport deficits. Together these data support that the RGC axonal projection remains at least semi-functional and structurally intact after anterograde transport loss in glaucoma. This pre-degenerative loss of transport may provide a target for innovative treatments aimed at restoring function in glaucomatous axons that still remain largely intact. ii Acknowledgments I would like to thank first and foremost my family for their love and support not only over the last two years, but throughout my life and academic career. I may never be able to repay the extent of everything they have done to guide my success in any of my endeavors, but I certainly will do my best. Secondly, I would like to acknowledge Dr. Inman for the tremendous steps she has taken to push the Integrated Pharmaceutical Medicine program forward and for being both a constant and patient mentor through this project. Your many insights have been indispensable. Additionally, I would like to thank my fellow graduate students Gina and Lucy for providing reassurance and comedic relief along this journey. Lastly, I would like to extend my greatest appreciation to Dr. Sam Crish and Dr. Christine Crish who have played and continue to play the most essential role in my development as a student and as a researcher. I am extraordinarily grateful for their mentorship that has reignited my passion for neuroscientific research, yet, has humbled me to know that I still have much more to learn. Most of all, the truly unique dynamic of the lab is a constant reminder that regardless of the path you take, what is most important, is that you enjoy doing it. iii Vita Research Technician, Northeast Ohio Medical University………………………………………….…2012 Teaching Assistant, Brain Mind and Behavior……………………………………………………….…..…2014 Fields of Study Major Field: Integrated Pharmaceutical Medicine Minor Field: Neurobiology iv Table of Contents Abstract ...............................................................................................................................................ii Acknowledgements...........................................................................................................................iii Vita ......................................................................................................................................................iv List of Figures ................................................................................................................................. viii General Introduction .........................................................................................................................1 Anatomical Orientation of the Eye .....................................................................................1 Primary Visual Pathway ......................................................................................................4 Axonal Transport..................................................................................................................5 Intraocular Pressure ............................................................................................................7 Neurobiological Deficits in Glaucoma................................................................................8 Transport Deficits in Glaucoma ....................................................................................... 10 Molecular Motor Implications ......................................................................................10 Metabolic Implications.....................................................................................11 Cytoskeletal Implications.................................................................................12 v Introduction to Methods ................................................................................................................ 15 Research Goals ................................................................................................................... 15 Mouse Model of Glaucoma................................................................................................ 15 Neuronal Tract Tracing..................................................................................................... 16 Pilot Trials........................................................................................................................... 18 Methods............................................................................................................................................ 19 Animals ............................................................................................................................... 19 Anterograde Transport Labeling (intravitreal injections) .......................................... 21 Retrograde Transport Labeling (intracranial injections into SC) .............................. 21 Tissue Collection and Preparation .................................................................................. 22 Immunohistochemistry for Structural Marker.............................................................. 22 Microscopy ......................................................................................................................... 23 Measurement of Retrograde Transport.......................................................................... 23 Measurement of Anterograde Transport and Structure .............................................. 24 Statistical Analysis and Variables.................................................................................... 24 Results .............................................................................................................................................. 26 FG Density Does Not Differ by Strain or Age in Control Mice...................................... 26 Bidirectional Tracing Main Effects and Interaction Results ........................................ 27 Bidirectional Tracing Does Not Differ Between D2G Controls and 3 mo. DBA/2J.... 29 Anterograde Tracing in the SC is Significantly Reduced at 9-months in DBA/2J ..... 29 Structure of SC Remains Intact Until 13 months of Age in DBA/2J ............................ 30 FG Density and Percent Are of Intact FG in Retina Are Correlated............................. 31 Retrograde Tracing in the Retina is Reduced at 9-months ......................................... 35 vi Structural Integrity of the SC is Maintained Beyond Retrograde Transport Loss .... 35 Discussion ........................................................................................................................................ 39 Structural Persistence of the SC....................................................................................... 40 Differential Transport Loss in Glaucoma ....................................................................... 40 Molecular Motor Modification and Transport Deficits................................................. 41 Metabolic Dysfunction and Transport Deficits.............................................................. 42 Cytoskeletal Alterations and Transport Deficits ........................................................... 43 Conclusion........................................................................................................................................ 45 References........................................................................................................................................ 46 vii List of Figures Figure 1: Anatomical Depiction of the Retina.......................................................................................3 Figure 2: Visual Pathway ...........................................................................................................................4 Figure 3: Neuronal Tract Tracing Scheme..........................................................................................17