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Mark K Webster, Cynthia Gossman, Cindy Linn; Proliferation of Retinal Ganglion Cells in a Mammalian Model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3615.
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© ARVO (1962-2015); The Authors (2016-present)
Preliminary studies from this lab have indicated that certain concentrations of the α7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, induces proliferation of adult rat retinal ganglion cells (RGCs). This study seeks to investigate the proliferation of RGCs in adult Long Evans rats in response to PNU-282987 eye drop treatment.
To label proliferating cells, an eye drop procedure was used twice per day that combined 2mM PNU-282987 and 1 mg/ml Brdu in PBS. In some animals, the right eyes of rats were treated with this mixture for 7 or 14 days. Animals were sacrificed, retinas were removed, fixed and double-labeled with anti-Brn3a and anti-Brdu. Left eyes were treated only with Brdu as an internal control. In other rats, the PNU-282987 and Brdu eye drop mixture was added to right eyes for 14 days but the animals weren’t sacrificed for another 14 days. Retinas were then processed as above. The number of double-labeled cells from experimental retinas were compared to the number of double-labeled cells from internal controls using 200 µm2 images obtained throughout the retina from a Nikon N2 confocal microscope. N’s of 4 were obtained for all experiments. Statistical analyses were performed using Student T-tests. P-values <0.05 were considered statistically significant.
When animals were treated with the eye drop mixture for 7 days and then sacrificed, an average of 6.5 ± 1.12 double-labeled cells were found in images obtained from the inner nuclear layer (INL), but no double-labeled cells were found in the retinal ganglion cell layer (GCL). This was significantly different from internal controls where no double-labeled cells were found under any experimental condition in any of the retinal layers. For animals treated with the mixture for 14 days and then sacrificed, there was an average of 13.5 ± 2.96 labeled cells in the 200 µm2 retinal image obtained from the INL, 3.25 ± 0.43 labeled cells in the IPL, but no labeled cells in the GCL. The animals treated for 14 days and sacrificed after another 14 days had an average of 11.25 ± 1.79 labeled cells in the 200 µm2 retinal image from the GCL, but no labeled cells in the IPL or INL.
These results suggest that proliferation of RGCs may be occurring after eye drop treatment using an α7 nAChR agonist in an adult mammalian system. As adult mammalian neurons do not typically reproduce, these findings have exciting and significant implications.
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