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K. S. Vidal, A. H. Kihara, E. S. Yamada, L. R. G. Britto; Distribution of Neurofilament Isoforms in the Human Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2144.
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Neurofilament proteins (NFPs) are found in the perikarya and especially in the axons of neurons throughout the central and peripheral nervous system. The purpose of this study is to characterize the distribution of neurofilament isoforms in human retinas by using immunohistochemistry technique
Human retinas were obtained from eyes previously used for cornea donation. Retinas were fixed in 4% paraformaldehyde in 0.1M phosphate buffer for 12h. Subsequently, retinas were dissected and the choroid and vitreous were removed. Retinal wholemounts were incubated in mouse anti-PAN antibody for 72 hours (1:1000) at room temperature, and wholemounts were rinsed and incubated with a biotinylated goat anti-mouse antibody followed by the avidin-biotin-horseradish peroxidase complex for 12h each. Immunoreactivity was revealed by the standard diaminobenzidine method. Anti-PAN neurofilament is a cocktail of three mouse monoclonal antibodies, which reacts with all three major polypeptides of human neurofilament, low (68 kD), middle (160 kD) and high (200 kD) molecular weight polypeptides. The number of labeled cells and their retinal distribution were established by using a Nikon microscope. We used a systematic sampling method to map the distribution along the entire retina at 2 mm intervals. A similar immunohistochemistry protocol to anti-PAN was performed to vertical sections. In addition, the vertical sections were also incubated in antibodies raised against specific 68kD, 160kD, 200kD neurofilaments (NF68, NF160, NF200).
Whole-mounts using anti-pan revealed a considerable number of fibers in the nerve fiber layer, a subset of large ganglion cells, and a population of smaller somas in the ganglion cell layer. These completely stained cells were detected in highest density values around the fovea. In central retina, near to the papilla of optic nerve, we could not observe a completely stained body cells, only axons and dendrites. Densities decreased concentrically towards the retinal periphery. In the vertical sections, the anti-NF200 revealed a population of large cells in the ganglion cell layer with the soma size six -fold larger than soma cells labeled by anti-NF68.
Our results suggest that at least two specific neurofilaments are expressed in distinct ganglion cells in the human retina. Since NFPs are major structural proteins, which are biochemically quite stable, antibodies raised against NFPs can be used in studies of neuronal expression, morphology, connectivity and pathology, like glaucoma.
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