Patterns of Expression of Neuroglobin and Cytoglobin in the Healthy and Glaucomatous Dog Retina

Abstract

Abstract: : Purpose: To determine the presence of neuroglobin (Ngb) and cytoglobin (Cygb) in healthy and glaucomatous dog retinas and their distribution in specific retinal cell types. Methods: Polyclonal anti–Ngb and anti–Cygb antibodies and a monoclonal anti–Ngb antibody were used to examine their patterns of distribution in dog retinal sections. Double–labeling studies were performed with antibodies against: MAP2,TUJ1, PKC α, calretinin, CRALBP and vimentin to examine the cellular distribution of Ngb and Cygb. Tissue sections were visualized and images captured with a confocal scanning laser microscope. Immunoblot analysis was performed to verify the specificity of the anti–Ngb and –Cygb antibodies. Results: Immunohistochemical analysis of dog retinal tissue showed Ngb immunoreactivity (–IR) in the retinal ganglion cell layer, inner nuclear layer, inner and outer plexiform layers and inner portion of photoreceptor outer segments. Ngb–IR was colocalized with MAP–2, TUJ1, PKC α and calretinin (neuronal markers), but not with CRALBP– or vimentin–IR cells (glial markers). Cygb–IR was found in the retinal ganglion cell layer, inner nuclear layer and inner and outer plexiform layers. Cygb–IR was colocalized with MAP–2, TUJ1, and PKC α but not with vimentin–IR cells. Ngb and Cygb were expressed in the same retinal layers, except the inner portion of photoreceptor outer segments where only Ngb was detected. Western blot analysis confirmed the presence of Ngb and Cygb in the dog retina. Ngb expression appeared to be increased in the inner retina of glaucomatous dogs. Conclusions: Ngb and Cygb, two recently discovered oxygen–binding globins, are widely distributed in dog retinal, but not glial cells. Due to structural similarity to hemoglobin and myoglobin, we hypothesize that Ngb and Cygb may have an important role in the retinal oxygenation during ischemic/hypoxic conditions and glaucoma.

Keywords: immunohistochemistry • ganglion cells • oxidation/oxidative or free radical damage