Imaging Apoptosis in the Living Eye: A Novel Method of Assessing Retinal Ganglion Cells and Glaucoma

Abstract

Abstract: : Purpose: Retinal ganglion cell (RGC) apoptosis is implicated in the pathogenesis of glaucoma. However, previously this has only been possible to demonstrate histologically. We present a novel method of assessing RGC apoptosis in vivo using a rat model. Methods: 10 DA rats underwent general anaesthetic following which varying doses of intravitreal staurosporine (0, 30, 60, 90, 120 ng) and a fluorescent-labeled annexin 5 marker of apoptosis (1.4 µg) were administered. Rat eyes were then imaged using confocal scanning laser ophthalmoscopy (cSLO) with an argon laser (488 nm) for illumination and a wide band-pass filter with short-wavelength cut-off of 521 nm filter. Videos of scanned retinal areas were recorded and images assessed for fluorescence using a previously established method. Animals were killed immediately after imaging, and enucleated eyes were fixed in fresh paraformaldehyde for histological analysis. Immunohistochemistry was performed using NF-200 antibody for retinal ganglion cells, and specimens were viewed by confocal microscopy. Results: Cells were seen to fluoresce in the retina with cSLO from 2 hours after treatment. Time-lapse video revealed a random distribution and appearance of fluorescent cells throughout the retina. These were confirmed histologically to be apoptotic retinal ganglion cells by double-labeling. Anatomical reconstructions of in vivo imaging and histological results showed a good correlation of methods. Eyes treated with staurosporine had, as predicted, a much higher number of apoptotic RGC, and staurosporine-induced RGC apoptosis was found to be dose-related. Conclusions: This study has shown that it is possible to image retinal ganglion cell apoptosis in vivo. Although we have used a powerful inducer of RGC apoptosis, we believe this method may be useful in assessing all forms of pathological retinal degeneration. Moreover, we feel this approach has great potential in the investigation and treatment of glaucoma. Acknowledgements: Roger Hitchings, Martin Wax, Len Levin

Keywords: ganglion cells • apoptosis/cell death • imaging/image analysis: non-clinical