Cytoarchitecture of the retinal ganglion cells in the rat

PURPOSE. To determine the number and cytoarchitecture of retinal ganglion cells (RGCs) in the female Wistar rat, by using a newly devised procedure for rapid RGC counting in the entire retina that avoids assumptions about RGC spatial arrangement. METHODS. RGCs of normal female Wistar rats were retrogradely labeled with a fluorescent tracer. Automated counting was accomplished by applying standard imaging software to analysis of all labeled cells in retinal flatmounts. The method was validated by comparison of automated and manual counts of 70,000 RGCs in frames covering the density range in the normal rat retina of 600 to 3600 RGC/mm 2 . RGC numbers were determined for each retina and compared with the contralateral retina of the same animal. RGC density maps were constructed for each retina. RGC size distribution was determined. RESULTS. Automated RGC counting showed a good linear correlation with manual counting (R 2 0.9416). Mean total RGC count in 10 rat eyes was 97,609 3,930 (SEM) per eye. Contralateral eyes differed by an average of 4.1% (3983 5098 RGCs). Size analysis calculated from cell areas confirmed that the majority of rat RGCs are between 7 and 21.5 m in equivalent diameter. The RGC counts for all frames at the same eccentricity in all 10 of the retinas showed that variability increased with eccentricity and increased further as the fractional area of the retina sampled at each eccentricity was reduced. There was also significant variability in the spatial density of the RGCs at the same eccentricity location between different eyes. Comparison of total RGC counts between left and right eyes estimated from RGC counts in sectors of the retina (hemiretinas or quadrants) showed increased variability compared with counting all the RGCs in a retina. CONCLUSIONS. RGCs in the Wistar rat display significant variability in their cytoarchitecture. Such variability can make quantification by sampling problematic for diffuse, and particularly, for focal RGC losses resulting from experimental interventions, unless virtually the entire RGC population is counted. (Invest Ophthalmol Vis Sci. 2002;43:587‐594) Q uantitation of retinal ganglion cell (RGCs) is a procedure that is often used to monitor the effects of noxious stimuli leading to cell death in the retina. This procedure has been applied to several experimental rodent retinal disease models, such as ischemia-reperfusion, 1 partial optic nerve crush, 2 optic nerve axotomy, 3,4 excitotoxin injection, 5 or chronic elevation of intraocular pressure (IOP). 6,7 The experimental eye is usually compared with the contralateral control eye for changes in the RGC number. Quantitation of RGC number has been performed by extrapolation from cell counts obtained from representative cross-sections, areas of retina in flatmounts, or by counting a proportion of the RGC axons in cross-sections of the optic nerve. Sampling methods are used in all these quantification procedures to overcome the difficulty of counting the large number of RGCs or their axons and the variations in cell or axon density. However, changes in the number of RGCs determined by comparison of sampled areas in retinal pairs has greatest reliability if it can be shown that the sampled areas from equivalent locations in the two retinas have equivalent RGC densities at the beginning of the experiment. There is a general radial symmetry in the spatial distribution of RGCs in the rat eye, with decreasing RGC density progressing from the optic nerve head to the retinal periphery. This symmetry is often assumed to be 360°, with the RGC density