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Y. Yaacobi, J. Yang, S. Cao, B. Li, M. Wax; Whole Mount Brn-3a Immunostaining of Mouse Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5472.
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Retrograde retinal ganglion cell (RGC) labeling is labor intensive and requires days to allow for axonal transport of tracers. Also, variable size and shape, clusters of stained RGCs, hazy retinal background, and sample bleaching make automated RGC counting impractical. The purpose of this study is to develop an ex vivo immunostaining method suitable for automated counting of RGCs in whole mount mouse retinas.
Brn-3a RGC Staining. Following perfusion, eyes (n=7 mice) were enucleated, fixated, anterior segment and vitreous removed, and whole retinas dissected. Remaining vitreous and inner limiting membrane were digested (plasmin), removed from the eye, RGC nuclei immunolabeled with Brn-3a (mouse to rat) antibodies, and automatically counted. Validation Studies: (1) Retinas (n=5 mice) were retrogradely labeled with Fluoro-gold (FG, 1µl of 4%), collected, 2-4 days post FG injections, and their RGCs semi-automatically counted in 4 central and 4 peripheral retinal areas. The total RGC population was then extrapolated and compared to the RGC count from the Brn-3a labeled RGC retinas (above) to determine the Brn-3a-labeled RGC subpopulation. (2) Eyes (n=44 mice) were intravitreally (IVT) injected, in one eye, with vehicle, 1, 5, 10, 20, 60, or 100 nmol N-methyl-D-aspartate (NMDA). Contralateral eyes served as controls. On day 7, eyes were harvested and retinas processed for Brn-3a RGC staining and counting.
FG retrograde labeled RGCs were shown to be in variable shape, size, and different levels of fluorescent brightness. The calculated average RGC population was 58,290 ± 5,345 (Mean ± SD, N=9). Brn-3a Stained RGCs exhibited dark brown, permanently stained, uniform nuclei, prominent on the homogeneous light brown retinal background. The average number of automatically counted RGCs, in naïve mouse eyes, was 30,236 ± 4,719 per retina (Mean ± SD, N=13). Compared to contralateral eyes, RGC count - Seven days post IVT injection of vehicle, 1, 5, 10, 20, 60, or 100 nmol NMDA / eye - respectively, showed 2%, 14%, 55%, 65%, 77%, 86% and 81% RGC loss.
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