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Invest Ophthalmol Vis Sci. 2013 Mar 21;54(3):2061-7. doi: 10.1167/iovs.12-11389.

Enrichment of retinal ganglion cells in rat retinal lysate by excimer laser ablation of the outer retina.

Author information

1
Department of Ophthalmology, Georg-August University Hospital Göttingen, Germany. christian.oterendorp@med.uni-goettingen.de

Abstract

PURPOSE:

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.

METHODS:

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).

RESULTS:

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 -iiitubulin (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).

CONCLUSIONS:

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.

PMID:
23449715
DOI:
10.1167/iovs.12-11389
[Indexed for MEDLINE]

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