Quantification of axotomized ganglion cell death by explant culture of the rat retina

Abstract

We first demonstrated a temporal profile of retinal ganglion cell (RGC) death after axotomy in situ using a newly developed retinal explant culture system. 1,1'- dioctadecyl- 3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent tracer, was administered to the superior colliculi of 2 day old Wistar rats to label RGCs retrogradely. Small pieces of retinas were dissected and maintained at the interface between a 5% CO(2) atmosphere and culture media, and temporally observed by fluorescent microscopy. The number of surviving RGCs, identified as fluorescent spots, gradually decreased during the course of experiments for up to 10 days in vitro. We identified apoptotic RGCs by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. Administration of cycloheximide, actinomycin D, or a caspase-3 inhibitor to media significantly decreased RGC death. This system provides a method of quantifying axotomized RGC death in relation to time-dependent changes in an identical retinal slip.