In Vivo Imaging of Retinal Ganglion Cell Axons within the Nerve Fiber Layer

PURPOSE. Optic nerve injury causes loss of retinal ganglion cells (RGCs) and their axons. The reduction in RGC counts over time in axonal injury is well studied, but the correlation with the timing of anterograde and retrograde axonal degeneration is less clear. The authors longitudinally imaged RGC axons stained with a chloromethyl derivative of fluorescein diacetate (CMFDA) in live rats after optic nerve injury. METHODS. Optic nerves were transected. Three days later CMFDA was intravitreously injected. Confocal scanning laser ophthalmoscopy was performed daily, and mean fluorescence intensity and the number of CMFDA bundles were calculated. RGC soma survival was studied after retrograde fluorescence labeling. Retinal nerve fiber layer (RNFL) thickness was evaluated histologically. RESULTS. CMFDA-positive RGC axon bundles could be imaged in vivo. Axons lost 68% +/- 29% of their fluorescence by 7 days after transection compared with 25% +/- 21% in nontransected eyes. The number of labeled axon bundles decreased by 61% +/- 28% at 7 days after transection compared with 26% +/- 9% in nontransected eyes. The number of retrograde-labeled RGCs detected in vivo declined by 53% at 7 days and by 76% at 14 days after transection. RGC soma and CMFDA axon counts decreased most rapidly between 5 and 7 days after transection. Histologic examination demonstrated a reduction in RNFL thickness 7 days after transection. CONCLUSIONS. Intravitreal CMFDA can be used to longitudinally monitor RGC axons within the RNFL in vivo. Imaging the disappearance of retrograde-labeled RGC somas and axons indicates that axonal and somal degeneration occur in parallel after axotomy. (Invest Ophthalmol Vis Sci. 2010; 51: 2011-2018) DOI: 10.1167/iovs.09-4021