In Sickness and in Health: Real–Time Assessment of Healthy and Diseased Retinal Ganglion Cells (RGCs) in vivo

Abstract

Abstract: : Purpose: Until now, objective measurements of RGCs in glaucoma have relied upon surrogate functional tests such as perimetry and electrophysiology, as it has not been possible to assess RGCs directly. We have recently developed a technique allowing direct visualization of RGC apoptosis in vivo. Validation of our results has relied on the histological demonstration of retrogradely–labeled RGCs. In this study, we investigate the possibility of assessing both apoptosing and intact RGCs in vivo and in real–time using different markers. Methods: We assessed rat models of glaucoma–related disease (chronic ocular hypertension and staurosporine–induced RGC apoptosis, n=30) in which either DiAsp, DiI or dextran rhodamine B (RhodB) were applied to the superior colliculus of animals before surgery. Animals were imaged repeatedly at baseline and regular intervals post–operatively with an adapted confocal scanning laser ophthalmoscope. Intravenous or intravitreal alexa fluor 488–labeled annexin 5 (annexin–488) was given at each time point to evaluate apoptosis, as previously described. Animals were killed at set times, and enucleated eyes analysed histologically. Results: The best images of RGC in vivo were achieved using RhodB. This marker also allowed us to simultaneously evaluate single RGC cells undergoing apoptosis in real–time, due to the distinct absorption/emission spectra of RhodB (570/590nm) and annexin–488 (495/519nm) fluorochromes. Good clear labeling was achieved with intravenous administration of annexin–488. Anatomical reconstructions revealed a good correlation of in vivo imaging with histological results, with confirmation of apoptotic RGC by double–labeling. Conclusions:We demonstrate the first direct visualization of simultaneously labeled intact and apoptosing single RGC cells in vivo. Although the demonstration of RGC apoptosis reflects disease activity at any one time, as RGC degeneration progresses, the extent of RGC loss becomes increasingly important. This study offers unique insights into the natural history of RGC loss and apoptosis in glaucoma, using for the first time, intravenous annexin–488, with obvious implications for clinical use. Our method provides a tool for objective measurement of RGC damage, as it occurs, and in longitudinal study, and is a clear example of a technique that promotes the 3Rs concept for animals in research.

Keywords: imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • apoptosis/cell death • ganglion cells