Every quadrant of the retina was analyzed separately. The automated Brn3a+ cell counting routines for Image-Pro Plus (Image-Pro Plus, IPP 5.1 for Windows; Media Cybernetics, Silver Spring, MD) for rat [24 (link), 40 (link), 41 (link)] and mouse retinas [25 (link), 42 (link)] were adapted to the analysis of chicken retinas. The chicken retinas are larger and contain in the order of 50 times more RGCs than a rat retina. Images were orderly selected from the reconstructed quadrant and converted to 16-bit gray scale, followed by the application of the higauss 7x7 enhancement filter. The resulting images were then filtered through a large spectral filter: Edge+, which extract positive edges from the dark background. Potential cell clusters were separated by the IPP watershed split morphologic filter. Cells were counted within predetermined parameters to exclude objects that are too large and too small to be the RGCs nuclei. Finally, data of each count were displayed and exported to a spreadsheet where they were filed and saved for further analysis. The total number of Brn3a+RGCs was obtained as the sum of the four quadrants of each retina.
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