In vivo imaging and counting of rat retinal ganglion cells using a scanning laser ophthalmoscope

PURPOSE. To determine whether a scanning laser ophthalmoscope (SLO) is useful for in vivo imaging and counting of rat retinal ganglion cells (RGCs). METHODS. RGCs of Brown Norway rats were retrogradely labeled bilaterally with the fluorescent dye 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodine (DiA). The unilateral optic nerve was crushed intraorbitally with a clip. RGCs were imaged in vivo with an SLO with an argon blue laser (488 nm) and optical filter sets for fluorescein angiography, before and 1, 2, and 4 weeks after the crush. Fluorescent cells were also counted in retinal flatmounts at baseline and 1, 2, and 4 weeks after the crush. An image overlay analysis was performed to check cell positions in the SLO images over time. Lectin histochemical analysis was performed to determine the relationship of microglia to the newly emerged DiA fluorescence detected by image overlay analysis after the optic nerve crush. RESULTS. Fluorescent RGCs were visible in vivo with an SLO. RGC survival decreased gradually after the crush. In the retina after the optic nerve crush, newly emerged DiA fluorescence detected by image overlay analysis corresponded to fluorescent cells morphologically different from RGCs in the retinal flatmount and was colocalized mostly with lectin-stained microglial processes. RGC counts by SLO were comparable to those in retinal flatmounts. CONCLUSIONS. The SLO is useful for in vivo imaging of rat RGCs and therefore may be a valuable tool for monitoring RGC changes over time in various rat models of RGC damage.