Characterizing the Effects of Experimental Glaucoma and Age on Retinal Ganglion Cell Function

Abstract

Glaucoma is an optic neuropathy characterized by the progressive loss of retinal ganglion cells (RGCs). Clinical monitoring of disease progression uses gross structural changes as surrogate measures of RGC loss. Previous studies indicate structural imaging relates poorly to RGC axonal counts, due to inter-individual variability, and nonneuronal tissue remodelling that occurs during glaucoma. Moreover, clinical imaging does not provide information on the functional status of individual cells. Therefore, there is a need for tools to monitor longitudinal structural and functional changes to individual RGCs. In this study, we delivered the functional marker, GCaMP, using an intravitreal injection of AAV2-CAG-GCaMP6s viral vector to evaluate RGC structure and function. GCaMP labelling, monitored with in vivo imaging, increased weekly over 5- weeks, and plateaued by 8-weeks. Results showed GCaMP labelling persists for over 18- months following a single viral injection. Intact-isolated calcium imaging demonstrated robust GCaMP functional responses to chemical stimulation in adult (3-6 months) and aged mice (21-24 months). When compared to adult mice, aged mice showed more profound decreases in functional responses. Immunohistochemistry confirmed that the AAV2-CAG-GCaMP6s viral vector primarily transduces RGCs in the ganglion cell layer, and does not negatively impact RGCs when compared to non-injected control mice. Although intraocular pressure and age are the most significant risk factors for glaucoma development and progression, most experimental glaucoma (EG) research is performed in adult mice. Therefore, for our study, EG was induced in both adult and aged mice. Functional responses and RGC densities were decreased compared to controls following 4-, and 8-weeks of EG in adult mice, and to a significantly greater extent in aged mice. Interestingly, functional decline preceded structural loss in both adult and aged EG. Overall, our findings demonstrate that exogenous functional markers can be used to assess structural and functional changes over time and that age is a vital determinant of loss of RGC function.