Publication Date

2014-02-20

Availability

Embargoed

Embargo Period

2016-02-20

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2013-07-24

First Committee Member

Richard K. Lee

Second Committee Member

Vance Lemmon

Third Committee Member

Kevin K. Park

Fourth Committee Member

Paul R. Thompson

Fifth Committee Member

Sanjoy K. Bhattacharya

Abstract

Glaucoma is the second leading cause of irreversible blindness worldwide and it is estimated that over 80 million people throughout the world will have glaucoma by the year 2020. Currently, increase intraocular pressure is the only known modifiable risk factor in glaucoma pathophysiology, and therefore the only treatment option available to patients and their physicians is to lower the eye pressure. This thesis work explores several approaches in understanding neurodegenerative retinal ganglion cell (RGC) death in the retina, and explores rhythmic stimulation as a potential therapeutic strategy toward neuroprotection of injured adult RGCs. We begin by exploring progressive changes in gene expression in the DBA/2J animal model of glaucoma utilizing traditional molecular biology techniques. Utilizing clinical imaging modalities we next explore in vivo kinetics of RGC loss in an optic nerve crush injury model. Finally, we assess rhythmic stimulation as a potential approach for neuroprotection of adult RGCs following optic nerve crush injury. Our central hypothesis is that replication of the rhythmic activity observed in developing RGCs will promote survival and axon regeneration of injured adult RGCs. To test this hypothesis we stimulate RGCs expressing a light-gated proton channel from the green algae Chlamydomonas reinhardtii. A Channelrhodopsin2 EYFP fusion protein is expressed exclusively by RGCs in Thy1-COP4/EYFP line 9 (ChR2-YFP) retinas. Using RGC specific Thy-1 driven expression of ChR2-YFP fusion protein, we both stimulate and track RGC survival over-time in vivo. Our results identify VGLUT2 and CHRNA6 as RGC markers whose expression is significantly reduced as DBA/2J mice develop glaucoma. We also identify the lack of a CNS-specific VGLUT3 isoform in the retina of DBA/2J animals. Clinical ophthalmoscopy adapted for the mouse eye allow us to follow RGC loss in vivo, comparing spectral domain optical coherence tomography (SD-OCT) and confocal scanning laser ophthalmoscopy (CSLO) imaging modalities to track the longitudinal progressive dynamics of RGC survival subsequent to optic nerve crush injury. We determine that a direct imaging approach utilizing CSLO in conjunction to RGC-specific EYFP expression results in stronger statistical power in tracking RGC loss than does utilizing nerve fiber layer (NFL) thickness by SD-OCT as a surrogate marker for RGC survival. Our data do not show statistically significant improvements in either SD-OCT derived NFL thickness or CSLO derived RGC density by following Channelrhopsin2 rhythmic stimulation therapy of injured RGCs. Although rhythmic stimulation did not provide a neuroprotective effect to injured RGCs in vivo, our results indicate that rhythmic stimulation is capable of restoring axon regenerative potential of adult RGCs. Retinas receiving rhythmic stimulation showed a modest increase in axons growing past the crush site, though not extending very far from the injury. Our future research direction will focus on likely mechanisms of stimulation dependent axon regeneration and potential combinatorial therapeutic options toward improving intrinsic survival of injured adult RGCs.

Keywords

regeneration; channelrhodopsin2; glaucoma; optogenetics; ophthalmology; stimulation

Share

COinS