A novel method for fluorescent labeling of postmortem porcine retinal ganglion cells

Purpose: The cause for retinal ganglion cell (RGC) death in glaucoma is unknown. Isolation of purified viable RGCs is an important tool for studying RGC cell biology. Studies involving retrogradely traced RGCs are typically performed by injecting the superior colliculus (SC) of a rodent with a fluorescent dye approximately 48 hrs before sacrifice. The objective of this study is to develop a novel technique for retrograde labeling of postmortem viable globes and subsequent isolation and culture of adult RGC in large mammals. Methods: Whole porcine globes obtained from a local abattoir were retrogradely labeled either by incubating 5 μM CellTracker CMFDA, or the carbocyanine dye DiD diluted in media on the optic nerve stump. Staining was verified by frozen cryosectioning followed by confocal microscopy. Cell viability was determined by incubating the entire retina with Celltracker CMAC following the retrograde incubation period. Results: Fluorescent labeling appears throughout the retinal ganglion cell layer. Cells appear viable throughout the neural retina up to 8 hours following retrograde labeling. Conclusions: The use of nontoxic fluorescent dyes is a novel technique for labeling retinal ganglion cells in postmortem eyes. This technique may facilitate labeling, isolation, and subsequent purification of viable postmortem retinal ganglion cells in higher mammals.