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Y. Yaacobi, S. Cao, B. Li, J. Yang, W. Gong, B. Li, M. Wax; A Novel, Automated, High Throughput, Retinal Ganglion Cell Counting System. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4380.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a high throughput, reproducible, retinal ganglion cell (RGC) counting system using a modified scanning system and Brn-3a immuno-labeled whole flatmount rat retinas.
Tissue Preparation. Whole retinas were dissected from rat eye cups (after two hour fixation in 4% paraformaldehyde), the vitreous and inner limiting membrane removed (to enhance antibody penetration), and RGC nuclei were labeled using Brn-3a (mouse to rat) antibodies. RGC Counting. A set of 2 to 120 glass slides was fed into a modified linear-array scanning system, which automatically scanned a seamless true color digital image of an entire retina and, using an optimized algorithm, automatically counted the RGC nuclei in the entire retinal image. Validation study. Fifteen Brown Norway rats, divided into 3 equal groups, underwent unilateral optic nerve transection (ONT) and were subjected to ipsilateral intravitreal injections (IVT, 5µl/eye) of either Brain Derived Neurotrophic Factor (BDNF, 1µg), combined BDNF (1µg) and Glial Derived Neurotrophic Factor (GDNF, 1µg), or vehicle immediately after ONT. The contralateral eye served as control. Animals were euthanized at 14 day post ONT/IVT and the eyes immediately enucleated for RGC immunolabeling using Brn-3a antibodies. The whole retina flat-mount slides were automatically scanned and RGCs counted using (a) the automated method (see above) and (b) a semi-automated method consisting of whole retinal image splitting into 7-8 strips whose RGCs were then counted with Image-Pro Plus (v. 5.1) software.
RGC nuclei, stained in permanent dark brown - black color, were prominent on the homogeneous background retina, depicting uniform shape and size. Overall scanning and counting time was 14 minutes per retinal slide. The average number of RGCs in the control eyes was found to be 86,007± 12,525 (Mean ± SD) by the semi-automated counting method, and 86,396 ± 6,206 by the automated system (P=0.836). The average RGC survival rate in eyes in the BDNF, BDNF+GDNF, or vehicle treated animals were, respectively, 59.4%, 79.9% and 26.2%, with the semi-automated method, and 56.6%, 69.1%, and 30.3% with the automated method.
A novel, high throughput, reproducible, retinal ganglion cell (RGC) counting system, using a modified scanning system, was successfully used to automatically count Brn-3a immuno-labeled whole flatmount rat retinas. The system is very useful for high throughput drug screening studies (e.g., glaucoma studies) requiring tissue processing and cell viability analysis.
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