Citation: Sparrow JR, Blonska A, Flynn E, et al. 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;54:281254: -282054: . DOI:10.1167 PURPOSE. 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.METHODS. Fundus autofluorescence (AF) images (558 lens, 488 nm excitation) were acquired in albino Abca4þ/À , and Abca4 þ/þ 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.RESULTS. The Bland-Altman coefficient of repeatability (95% confidence interval) was 618% 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 À/À mice than in Abca4mice 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 À/À 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 À/À mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age.CONCLUSIONS. 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.Keywords: Abca4, RPE lipofuscin, quantitative fundus autofluorescence, mouse, bisretinoid F undus autofluorescence (AF) imaging using a confocal scanning laser ophthalmoscope (cSLO) is a noninvasive approach to monitoring the natural autofluorescence of the retina generated from the bisretinoids of lipofuscin in RPE. In early studies of fundus AF in human subjects, quantitation at discrete positions on the fundus was done by noninvasive spectrophotometry. [1][2][3] This work contributed to our understanding of the relationship between RPE lipofuscin accumulation and age, and demonstrated increases in fundus AF in retinal disorders, such as recessive Stargardt disease, 3,4 caused by mutations in the ATP-binding cassette (ABC) transporter ABCA4.5 Conversely, imaging of fundus AF by cSLO records the spatial distribution of fundus AF [6][7][8][9] and is valuable as a means to monitor specific patterns of AF in human retinal diseases, including age related macular degeneration and RP.9-11 Some studies have reported comp...

In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

Vitrectomy with induction of PVD is a safe and successful therapeutic option for the treatment of optic disc pit maculopathy. The adjunct of ocriplasmin might facilitate the induction of PVD and reduce the risk of iatrogenic retinal holes.