Quantitation of Mouse RGCs by Direct Labeling With Antibodies to Neurofilament, PGP 9.5, Osteopontin, and Brn3 Compared to Retrograde Labeling With Aminostilbamidine

Abstract

Abstract: : Purpose: To evaluate alternative methods of assessing retinal ganglion cells (RGCs) in pathological retinas. To develop a direct labeling method for quantifying RGCs in flat–mounted mouse retinas without in vivo pre–labeling of RGCs with retrograde tracers but that can be combined with such labeling. To quantitatively compare retrograde Aminostilbamidine (AS) labeling with immunolabeling of RGCs using antibodies against Neurofilament–200 (NF), Protein Gene Product 9.5 (PGP 9.5), Osteopontin (OPN) and Brn3. Methods: Flat–mounted retinas were prepared after perfusion–fixation with either 2% or 4% paraformaldehyde. Optimal staining conditions were first determined for NF, PGP 9.5, OPN and Brn3 alone on retinal whole mounts. For subsequent testing for OPN and Brn3 immunolabeling, retinas were also pre–labeled with AS by prior application of the tracer to the superior colliculus one week earlier. AS fluorescently tagged and OPN or Brn3 immunolabeled retinas were digitally imaged. The number of dual–labeled RGCs was assessed in random images at 560X magnification using image analysis software. Results: In initial experiments, antibodies to NF and PGP 9.5 stained RGCs, but also axons and dendrites. Anti–NF antibody stained more RGCs and also more intensely than the anti–PGP 9.5 antibody. Both anti–OPN and anti–Brn3 antibodies stained RGCs with little labeling of axons and dendrites. On double labeled retinas, 98± 4.7% of AS labeled RGCs were also labeled for OPN, while less than 1% of OPN labeled RGCs were not labeled for AS. A total of 65.9 ± 19.7% AS labeled RGCs were also labeled for Brn3, while 8.8 ± 4.7% of Brn3 positive cells were not labeled with AS. The Brn3 labeling of RGCs was non–uniform, either intense (38.8 ± 8.1%) or weak (61.2 ± 8.1%). Conclusions: Anti–NF and anti–PGP 9.5 antibodies label RGC axons and dendrites, and are thus not suitable for automated image analysis to quantify RGCs. Immunolabeling of RGCs by anti–OPN antibody is equivalent to AS labeling. Antibody to Brn3 labels subgroups of RGCs including some not labeled by AS. Antibodies to OPN or Brn3 can be used as alternatives to AS labeling or combined with such labeling to distinguish between RGCs with functional and damaged axons.

Keywords: ganglion cells • immunohistochemistry