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K. Stecher, A. Messias, A. Baryluk, B. Prayon, E. Zrenner, F. Gekeler; Recovery of the A- and B-Wave of the ERG After Photo Pigment Bleaching in Rats Using a Modified Double Flash Paradigm to Assess Visual Cycle Function. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1294. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To define optimal ERG protocol parameters practicable for monitoring rhodopsin recovery after photo pigment bleaching.
First, in 51 wild-type brown-Norway rats, VMax , k, and n of the Naka-Rushton function were derived using stimuli of increasing luminance in a range of 6 logs. Second, a paired flash paradigm (test flash: 3, probe flash: 30 cd.s/m²) at inter stimulus intervals from 200-4000 ms was applied to evaluate a-wave recovery after rod desensitization (bleaching<0.5%). Finally, recovery of rhodopsin was evaluated every 2.5 min up to 70 min after light exposure using a modified double flash protocol that elicited a semi-saturated rod b-wave (flash α: 0.003 cd.s/m²) and after 1 sec a saturated a-wave (flash ß: 10 cd.s/m²). Rats were assigned to 6 groups according to bleaching level: (A) 2.5 cd/m² for 25 sec, (B) 10 cd/m² - 25 sec, (C) 100 cd/m² - 25 sec, (D) 250 cd/m² - 25 sec, (E) 400 cd/m² - 30 sec and (F) 500 cd/m² - 60 sec. A, B, C, D: n=8; E: n=38 (13 were examined twice for assessment of repeatability); F: n=5. At last, all animals were light adapted 10 min with 25 cd/m² for cone responses.
Parameters of the Naka-Rushton function were VMax: 873.4 ± 188.7 µV, k: -3.21 ± 0.25 log cd.s/m², n: 0.99 ± 0.06 (n=51; mean ± SD). The test flash abolished the probe a-wave completely at 200 ms (ISI), recovery to half of maximum was found at 1267.78 ± 17.09 ms. After bleaching, in group A the b-wave elicited by flash α had an estimated half maximum amplitude after 2.4 ± 0.09 sec in the dark; in B: 1.16 ± 0.41 min; in C: 3.52 ± 0.37 min; in D: 17.54 ± 0.36 min (repeatability coefficient: 2.SD = 2.60 min); E: 24.74 ± 0.67 min; and in F: 70.37 ± 2.25 min. The a-wave elicited by flash ß showed faster recovery with half maximum in D at 5.83 ± 0.29 min; in E at 10.73 ± 0.14 min; and in F at 33.19 ± 0.92 min. Cone single flash b-wave amplitude was 105.22 ± 3.29 and flicker amplitude 18.68 ± 0.29 µV.
Selection of the optimal parameters in conjunction with strict adherence to the protocol yields low intra- and inter-individual variability in all steps. Since rhodopsin recovery depends on the integrity of the photoreceptor-RPE interaction the procedure is apt to assess visual cycle function in retinal degenerations and test influences of various drugs and toxins (e.g. retinoids, heavy metals).
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