Abstract
Abstract: :
Purpose:Vision loss in glaucoma is attributed to retinal ganglion cell (RGC) apoptosis. However, until now this process has only been demonstrated histologically. Unlike most other organs in the body, direct visualization of retinal disease mechanisms in the eye is possible because of clear optical media. Using a novel method of assessing RGC apoptosis in vivo, we investigated RGC apoptosis in several glaucoma–related models of RGC disease. Methods:Models of chronic ocular hypertension (OHT, 20 DA rats), optic nerve transection (18 DA rats), and a newly–developed model of staurosporine–induced RGC apoptosis (15 DA rats and 2 macaque primates), were assessed whilst under anaesthesia. Intravitreal fluorescent–labeled annexin 5 was given immediately before imaging with an adapted confocal scanning laser ophthalmoscope (cSLO), as previously described. Animals were killed at set times, and enucleated eyes analysed histologically. Results:We observed single RGC cells undergoing apoptosis in vivo, in 3 different models. In axotomised eyes, we recorded apoptosis in 0.3%, 1%, 8% and 3% of total RGCs, with RGC losses of 0, 3%, 40% and 76% at 0, 3, 7 & 12 days respectively, and in 1%, 15%, 13%, 7% and 2% of total RGC, with RGC losses of 17%, 22%, 36%, 45% and 60% of the original population at 2, 3, 4, 8 & 16 weeks respectively of OHT eyes. Anatomical reconstructions revealed a good correlation of in vivo imaging with histological results, with confirmation of apoptotic RGC by double–labeling with DiAsp and anti–Caspase–3. Staurosporine was found to induce dose–dependent effects. There was a significant positive correlation of RGC apoptosis with ΔIOP integral (r 2 = 0.89, p<0.01) in OHT eyes after 8 weeks IOP elevation. Conclusions:We demonstrate the first direct visualization of single cells undergoing apoptosis in vivo, and in experimental glaucoma. We record changes over time with peak apoptosis occurring at 7 days post–axotomy and 3 weeks after IOP elevation. Effects appear dependent on the magnitude of the initial insult. These exciting results suggest that this novel technique may be important not only in glaucoma, but also other ocular disorders in which apoptosis is implicated, where, in addition to monitoring disease activity and treatment efficacy, it may provide an alternative and sensitive tool for the early detection of abnormalities.
Keywords: apoptosis/cell death • imaging/image analysis: non–clinical • ganglion cells