Enucleation was necessary to calculate the RGC change in density, as each J:DO mouse has a unique genetic constitution
23,38 and RGC number is genetic background dependent.
39 Thus, it was expected that the two eyes of an individual mouse would have a similar number of RGCs (because they share the same genetic background) but differ between animals (because outbred mice have differing genetic backgrounds), making it necessary to consider RGC parameters on a per mouse basis. Four weeks following blast exposure, mice were euthanized, whole eyes were enucleated, the posterior cups dissected and fixed for a total of four hours in 4% paraformaldehyde. The immunohistochemical labeling of BRN3A-positive cells was performed as previously described.
40 Briefly, the posterior cups were incubated in a 0.3% Triton-X100 solution in phosphate buffered saline (PBST) overnight at 37°C, and retinas were dissected and bleached in a 3% hydrogen peroxide solution in 1% sodium phosphate buffer for three hours at room temperature. Retinas were permeabilized for 15 minutes at –80°C in PBST, blocked in a 2% normal donkey serum in PBST overnight, immunohistochemically labeled using an anti-BRN3A antibody (1:200; sc-8429; Santa Cruz Biotechnology) in 2% normal donkey serum, 1% Triton-X 100, and 1% DMSO at 4°C for two nights, followed by incubation with a secondary antibody (1:200, Alexa Fluor 488 donkey anti-goat, Invitrogen) for four hours at room temperature, counterstained with TO-PRO-3 Iodide (1:1000, Molecular Probes), transferred to glass microscopy slides, and flat-mounted using ProLong Diamond Antifade Mountant (Fisher Scientific) and cover-slipped. Flat-mounted retinas were imaged by confocal microscopy (Zeiss LSM 710) at a total magnification of 400×. For each retina, four confocal images were collected (1024 × 1024 px, 0.18 mm
2 image area) from nonoverlapping fields in the central retina, with z-stacks of three to five images collected for each image. Images were collected from the central retina adjacent to the optic nerve head (for diagram defining the central retina, see
Fig. 1 in Hedberg-Buenz et. al.
40). Images of BRN3A-labeled nuclei were processed in ImageJ, by first z-projecting at maximum intensity, followed by the
Subtract Background tool with rolling ball radius set to 35 pixels, followed by the
Smooth tool. Images were then converted to binary using Huang thresholding. Binary images were further processed using the
Open,
Watershed, and
Fill Holes functions. To count BRN3A-positive cells, the
Analyze Particles function was applied to the BRN3A images with particle size set to 20 to 150 µm
2 and circularity 0 to1. Only retinas that were isolated without dissection-induced damage, such as large tears, and were fully intact were used for the purpose of this study.