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M. Salinas–Navarro, S. Mayor–Torroglosa, T. Holmes, A. Ortiz, J.M. Bernal, I. Canovas, M.E. Aguilera, R.D. Lund, M.P. Villegas–Pérez, M. Vidal–Sanz; Automatic Quantitative Analysis of Retinal Ganglion Cells That Project to the Superior Colliculi in Adult Sprague–Dawley Rats . Invest. Ophthalmol. Vis. Sci. 2005;46(13):271.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To quantify the population of rat retinal ganglion cells that project to both superior colliculi (SCi), their main target territory in the brain. Methods: Adult female SD rats were anaesthetized and retinal ganglion cells (RGC) were retrogradely labelled with 3% Fluorogold (FG) applied over the surface of both SCi. One week later the rats were perfused, their retinas dissected and prepared as whole–mounts. Retinas were examined and photographed under a fluorescence microscope (Axioscop 2 plus, Zeiss, Germany) equipped with a digital high–resolution camera (ProgRes c10, Jenoptik, Jena, Germany) and a motorized stage connected to an image analysis system (Image–Pro Plus (IPP), ver. 184.108.40.206 and a pluging Scope–Pro, ver 5.0; MediaCybernetics, Inc. USA). The retina was imaged by adjacent, nonoverlapping frames captured in raster pattern (154 frames per retina) measuring each 0.6274 mm2 at a resolution of 0.8345 pixels/mm2. Each image was converted into 8 bits gray scale, flatten filter was used to reduce the intensity variations in the background pixels and a large spectral filter was used to increase the cells quality and maximize resolution of touching cells. Impulse noise was removed with a median filter. Adjacent cells were separated using the watershed split function and counted. The areas of these retinas were measured from photomontages with the aid of the tiling function of the IPP. To compare with the automated counts, manual counts of 35000 FG–labelled RGCs, in randomly selected digital images from 4 normal retinas, were performed by 3 different experimented investigators in a blind masked fashion. Results:One week after FG application to both SCi the total number of RGCs was 84,895±3,016 (mean ±SD) (n=11). These retinas had a mean area of 49.65±2.09 mm2 and this yielded a mean (±SD) density of 1,713±99 (n=11) FG–labelled RGCs per mm2. Manual counts of 35,000 FG–labelled RGCs produced similar densities, and showed a linear correlation with automated counts of r2 = 0.98. Conclusions: Using IPP the population of RGCs retrogradely labelled with FG from the SCi may be counted automatically with a level of confidence that is comparable to that found when RGCs are counted manually. Moreover, these results compare favorably to those obtained previously by other authors using similar methodology in female Wistar rats (Danias et al., 2002; IOVS 43:587).
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