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

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


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.

MeSH terms

  • Animals
  • Blotting, Western
  • CREB-Binding Protein / metabolism
  • Laser Therapy / methods*
  • Rats
  • Retina / surgery
  • Retinal Ganglion Cells / chemistry*
  • Rhodopsin / metabolism
  • Thy-1 Antigens / metabolism
  • Tubulin / metabolism


  • Thy-1 Antigens
  • Tubulin
  • beta3 tubulin, mouse
  • Rhodopsin
  • CREB-Binding Protein