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Christian van Oterendorp, Wolf A. Lagrèze, Eva-Maria Gutekunst, Stavros Sgouris, Philip Maier, Julia Biermann; Enrichment of Retinal Ganglion Cells in Rat Retinal Lysate by Excimer Laser Ablation of the Outer Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(3):2061-2067. doi: 10.1167/iovs.12-11389.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGC) are a relatively small cell population in the retina. This leads to an unfavorable signal-to-noise ratio when analyzing 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 before lysation.
Outer retinal layers were ablated with an excimer laser on flat mounted retinas from adult albino rats. 4′6-Diamidino-2-phenylindole dihydrochloride hydrate (DAPI) nuclear staining was used to assess the ablation efficacy (n = 6). Western blot for layer specific markers (rhodopsin, parvalbumin, β-III-tubulin) was performed to quantify changes in protein composition (n = 7). Excimer-ablated (EX) and full-thickness (FT) retinas 48 hours after optic nerve crush (ONC) were compared regarding the effect on phospho–cAMP response element binding protein (pCREB) and Thy1 levels (n = 5).
Area quantification of DAPI-stained retinas showed that 73% ± 4.9% of the ablation area was free of photoreceptor and bipolar cell nuclei. In Western blot, laser ablation led to a significant reduction of the photoreceptor marker rhodopsin and increase of the ganglion cell layer (GCL) marker β-III–tubulin (relative quantity: rhodopsin 0.47 ± 0.05, P < 0.0001; β-III–tubulin 2.35 ± 0.37, P = 0.02). Changes of pCREB and Thy1 after ONC were significantly different between FT and EX retinas (relative quantity pCREB: FT 1.4 ± 0.16, EX 0.78 ± 0.07, P = 0.008; Thy1: FT 0.95 ± 0.02, EX 0.63 ± 0.07, P = 0.006).
We demonstrated that excimer laser ablation of outer retinal layers is feasible, producing RGC-enriched retinal lysate. Laser ablation may allow a more specific detection of RGC responses to experimental stimuli.
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