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J. Kwong, J. Caprioli, N. Piri; Quantification of RNA Binding Protein With Multiple Splicing, Rbpms, as a Retinal Ganglion Cell Marker. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3090.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the expression of the RNA binding protein, Rbpms, in the rat retina as a specific marker for retinal ganglion cells (RGCs).
Adult male Wistar rats weighing 300-350g were used to examine the expression of retinal Rbpms. In situ hybridization with sense and anti-sense riboprobes (n=4) and RT-PCR (n=4) were performed on retinas 2 weeks after optic nerve axotomy. Antibody was generated against 22 aa polypeptide GGKAEKENTPSEANLQEEEVR located in the N-terminal of the protein. Whole retina after retrograde labeling with Fluoro-gold (FG) was obtained for immunhistohistochemistry and flat preparation (n=4). The numbers of RGC labeled by FG and Rbpms at locations from posterior to peripheral retina were quantified. To compare with other RGC markers, double fluorescence immunohistochemistry with antibodies against Thy-1/beta III tubulin/neurofilament and Rbpms were performed on FG labeled retinal sections (n=6).
In situ hybridization signals of Rbpms were predominantly localized in cells of the RGC layer and co-localized with FG labeling. Two weeks after optic nerve axotomy, RT-PCR showed a dramatic decrease in Rbpms mRNA levels in retina and in situ hybridization demonstrated a marked reduction of signal in the RGC layer compared to controls. Quantitative analysis of flat mounted retinas showed that the numbers of FG positive cells per mm2 were 2691±76, 2554±54, 1858±251 and 1377±305, and the numbers of Rbpms positive cells per mm2 were 2744±86, 2603±87, 1911±264 and 1418±326 at the distance of 1, 2, 3 and 4 mm from the center of optic nerve head, respectively. More than 99.5% of FG positive cells were Rbpms immuno-positive. Counting of immuno-positive cells in retinal sections demonstrated that 96.7±2.0%, 94.4±1.8% and 95.7±2.6% of FG positive cells were double immuno-labeled by Rbpms and beta III tubulin, neurofilament and Thy-1, respectively.
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