Retinal Ganglion Cells In Adult Albino And Pigmented Rats: Spatial Distribution And Quantitative Analysis.

Abstract

Purpose: : To analyze the spatial distribution and to quantify the population of retinal ganglion cells (RGCs) in adult rats.

Methods: : In adult (180–200 g) albino Sprague Dawley (SD) (female, n=45) and pigmented Piebald Virol Glaxo (PVG) Brown Norway (male and female, n=11) rats, RGCs were back–labelled with 3% Fluorogold (FG) in 0.9% NaCl applied to both superior colliculi (SCi) (n= 42) or to the intraorbital portion of the optic nerve (ON) (n=14). One week later rats were perfused, the retinas prepared as whole–mounts and examined and photographed under a fluorescence microscope (Axioscop 2 plus, Zeiss, Germany) equipped with a digital camera (ProgRes c10, Jenoptik, Germany) and a motorized stage, connected to an image analysis system with an automatic frame–grabber device (Image–Pro Plus, ver. 5.1.2) and a plug–in Scope–Pro, ver 5.0 (MediaCybernetics, Inc. USA). Retinas were imaged as a stack of 24–bit color images (154 frames per retina) using image–pro with the scope–pro plug–in 5.0; these images were montaged into a composite high resolution retinal map when required. Images are processed by specific macros written in IPP that apply a sequence of filters and transformations to each image of the stack in turn before finally counting the resulting cells and transferring the data to a spreadsheet for analysis. RGC densities were calculated from each frame by dividing RGC counts by the area of retinal tissue in the frame. Image analysis software (Photoshop) was used to generate pseudocolor density maps for each retina.

Results: : One week after FG application to both SCi the total number of RGCs in SD rats (81,486±4,339; n=37) was significantly smaller (Mann Whitney test, p=0.001) than in PVG rats (84,597 ±1,960; n=22). In SD rats, the total number of FG–labelled RGCs from the SC was comparable (Mann Whitney test, p=0.35) to that obtained labelling the ocular stump of the intraorbitally transected ON for 5 days (82,818±3,949; n=14). RGC densities were higher in the central retina and pseudocolor density maps revealed the presence in the superior retina of a region with the highest RGC densities. This region was located 1–2 mm dorsal to the optic disc and formed a low curvature arc that resembled a visual streak.

Conclusions: : SD and PVG rats show an arciform area in the superior retina that contains the highest RGC densities.

Keywords: ganglion cells • image processing • imaging/image analysis: non-clinical