Evaluation of Fluoro-Jade C as a marker of degenerating neurons in the rat retina and optic nerve

Abstract

Detection of neuronal death is an essential requirement for researchers investigating retinal degeneration. Fluoro-Jade C (FJC) is a novel, fluorescent dye that has been successfully used to label degenerating neurons in the brain, but its effectiveness in the eye has not been ascertained. In the current study, we determined the efficacy of FJC for detection of neuronal degeneration in the retina and optic nerve in various paradigms of injury. N-methyl-D-aspartate (NMDA) and kainic acid-induced excitotoxicity, optic nerve transection, and bilateral occlusion of the common carotid arteries (BCCAO) were performed using standard techniques. Rats were killed at various time points and the retinas with optic nerves attached were removed for tissue processing prior to labelling for FJC, for DNA fragmentation by TUNEL or for immunohistochemical analysis. Retinas from RCS rats of different ages were also analysed. After excitotoxicity-induced injury, cell bodies and dendrites within the ganglion cell and inner plexiform layers were specifically labelled by FJC within 6h, a time point comparable to the appearance of TUNEL-positive nuclei and to reductions in mRNA levels of retinal ganglion cell-specific proteins, but in advance of alterations in some immunohistochemical markers. The number of FJC-labelled cell bodies in the retina declined over time as cell loss proceeded, although dendritic staining remained prominent. Colocalisation of FJC with TUNEL and with immunohistochemical neuronal markers was achieved. FJC was successful at identifying somato-dendritic degeneration following ischemia induced by BCCAO, but surprisingly, not after optic nerve transection. FJC visualised photoreceptor degeneration in the RCS rat, albeit less effectively than with the TUNEL assay, and was also effective for imaging and quantifying degenerating axons in the optic nerve after multiple injuries. In addition to labelling degenerating neurons, however, FJC also bound non-specifically to astrocytes and to blood cells in unperfused rats. Since the ganglion cell layer is adjacent to astrocytes within the nerve fibre layer, caution is needed when using FJC as a quantitative tool for detecting ganglion cell death.