Quantitative Fundus Autofluorescence in Mice. Correlation with HPLC Quantitation of RPE Lipofuscin and Measurement of Retina Outer Nuclear Layer Thickness

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 582 ipofuscin • 551 imaging/image analysis: non-clinical • 688 retina