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X. Wang, M. L. Archibald, B. C. Chauhan; Cyan Fluorescent Protein (CFP) Expressing Cells in the Retina of Thy1-CFP Transgenic Mice Before and After Optic Nerve Injury. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2780.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the specificity of cyan fluorescent protein (CPF) expressing cells to retinal ganglion cells (RGCs) of the transgenic Thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) mouse line, and the characteristics of these cells after optic nerve injury.
RGCs of adult Thy1-CFP mice were retrogradely labeled with fluorochrome (2% fluorogold [FG]) from the superior colliculi (SC). Animals (n = 4) were sacrificed 7 days after RGCs labeling. Retinas were fixed and whole-mounted. CFP and FG positive cells were visualized and imaged using XF114-2 and UV-2A filters separately. Cells positive for either CFP or FG, or co-labeled were counted using the Infinity Analyze software. In another group of animals, the left optic nerves were transected at approximately 0.5 mm behind the globe 7 days after FG labeling. Animals were sacrificed at 7 and 21 days (n = 2 for each time point) after transection, retinas were whole-mounted and the characteristics of CFP expressing cells were examined.
CFP expressing cells were distributed evenly in the retina of Thy1-CFP mice. The average densities of CFP and FG positive cells in the retina were 2778 ± 216 and 3230 ± 157 cells/mm2 respectively. 93.2 ± 1.6% of CFP expressing cells were also labeled with FG. However, only 79.9 ± 2.5% of FG labeled RGCs expressed CFP. The number of CFP expressing cells decreased dramatically at 7 days after transection, and only a few CFP expressing cells and axons were observed at 21 days. Spindle shaped cells, presumably microglia, were seen in the retina with CFP expression at both 7 and 21 days after optic nerve transection.
The Thy1-CFP transgenic mouse is a powerful tool for the analysis of RGC survival in vivo since the large majority of CFP positive cells are RGCs. However, a large minority of RGCs do not express CFP. Furthermore, after injury to RGCs, CFP is internalized by phagocytosing cells potentially leading to inaccuracies in RGC estimates when counts of CFP positive cells are made.
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