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Janet Sparrow, Anna Blonska, Erin Flynn, Tobias Duncker, Jonathan Greenberg, Roberta Secondi, Keiko Ueda, Francois 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(15):4871.
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Quantitative fundus autofluorescence (qAF) analysis offers a non-invasive approach for measuring the natural autofluorescence of bisretinoid lipofuscin in the retinal pigment epithelium (RPE). This study sought to establish a standardized method for qAF analysis in Abca4 null, heterozygous, and wild type mice.
Fundus AF images (55° lens; 488 nm excitation) were acquired in albino Abca4-/-, Abca4+/- and Abca4+/+ mice (ages: 2-15 months) with a confocal scanning laser ophthalmoscope (cSLO). The cSLO was modified by insertion of an internal fluorescent reference to compensate for changes in laser power and detector gain, and custom-made apertures were used to reduce the laser beam diameter in proportion to the mouse pupil. Each image was a mean of nine frames without histogram stretching, and was analyzed with a dedicated image analysis program. Mean grey levels (GLs) from 8 pre-defined segments of the fundus were recorded. qAF was determined by calibrating the average GL of all segments to that of the reference by a factor of 0.7, after correction of the zero GL. The bisretinoid A2E was measured by quantitative HPLC. Histometric analysis of ONL thickness was performed to assess photoreceptor cell viability.
qAF increased with age (2-12 months) in all genotypes. qAF was 1.8 to 2.6 fold-higher in Abca4-/- than in Abca4+/+ mice and ~15% higher in heterozygous mice. HPLC measurements of A2E revealed age-associated increases, but the difference between Abca4-/- and wild-type mice was more pronounced (3-4 fold) than measured by qAF. Moreover, A2E levels declined after 8 months of age in Abca4-/- mice, a change not observed in qAF. A reduction in A2E was not observed in Abca4+/- and Abca4+/+ mice. This decline corresponded to reduced photoreceptor cell viability, as reflected in ONL thinning beginning at 8 months of age in Abca4-/- mice.
This qAF method enables measurement of in vivo lipofuscin and detected genotype and age-associated differences. This approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.
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