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Janet R. Sparrow, Anna Blonska, Erin Flynn, Tobias Duncker, Jonathan P. Greenberg, Roberta Secondi, Keiko Ueda, François C. Delori; Quantitative Fundus Autofluorescence in Mice: Correlation With HPLC Quantitation of RPE Lipofuscin and Measurement of Retina Outer Nuclear Layer Thickness. Invest. Ophthalmol. Vis. Sci. 2013;54(4):2812-2820. doi: 10.1167/iovs.12-11490.
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Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age.
Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4 −/−, Abca4 +/−, and Abca4 +/+ mice (ages 2–12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed.
The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2–12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4 −/− mice than in Abca4 +/+ mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4 −/− and wild-type mice was more pronounced (approximately 3–4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4 −/− mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age.
The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.
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