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Christian van Oterendorp, Wolf A. Lagreze, Philip Maier, Julia Biermann; Enrichment of Retinal Ganglion Cells in Retinal Lysate by Excimer Laser Ablation of the Outer Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3491.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells are a relatively small cell population in the retina. This leads to an unfavorable signal to noise ratio when analysing RGC proteins in whole retina lysate. We present a novel technique to obtain RGC enriched rat retinal lysate by removing the outer retinal layers with an excimer laser prior to lysation.
Flatmounted retinae from adult albino rats were fixated with methanol (n=4). The outer retinal layers were ablated with an excimer laser for clinical use (Schwind Amaris, Germany) over a diameter of 7 mm. The non-treated peripheral retina was removed and the remaining inner retina was either processed for microscopy or lysed for western blotting. Microscopic images with DAPI stained nuclei were used to measure the size of the remaining inner nuclear layer islets. Western blot for layer specific markers (rhodopsin for photoreceptors; Thy1 for RGCs) was used to quantify the effect of the ablation.
Morphological analysis of four treated retinae showed that the ganglion cell layer remained intact in 74 +/- 2.4 % (mean and SEM) of the total treated area. The remaining islets of the inner nuclear layer comprised 11 +/- 3.5 % of the GCL area. In western blots no rhodopsin was detected in any ablated retina while Thy1, normalised with Actin, was increased.
As a proof of principle we demonstrate that excimer laser ablation of outer retinal layers is feasible. The photoreceptors can be completely removed. The inner nuclear layer can at least be reduced in thickness and the retinal lysate can be used for western blotting.
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