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

Abstract

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...

Photodegradation of retinal bisretinoids in mouse models and implications for macular degeneration

“…For example, A2E levels in the vitamin E-treated mice were 54% higher (P < 0.05). Similarly, noninvasive in vivo measurement of RPE lipofuscin by quantitative fundus autofluorescence (qAF) analysis (16), revealed higher levels of fundus autofluorescence in mice treated with vitamin E from age 1-8 mo (P < 0.05) (Fig. 4C).…”

“…Quantitative Fundus Autofluorescence. Images were acquired and analyzed as previously described (16).…”

“…4C). Importantly, the thinning of ONL that is indicative of reduced photoreceptor cell viability in albino Abca4 −/− mice (15,16,31) was less pronounced in the vitamin E-treated mice (Fig. 4D).…”

“…Marked bisretinoid accumulation is replicated not only in Abca4 null mutant mice (9,10) but also in mice deficient in retinol dehydrogenase-8 and dehydrogenase-12 (Rdh8; Rdh12) (11)(12)(13). In addition, the relationship between RPE lipofuscin accumulation and retinal disease is evidenced in the Abca4 mouse model by complement dysregulation, carbonyl-deposition, thickening of Bruch's membrane, and a progressive loss of photoreceptor cells (11,(14)(15)(16)(17).…”

Therapeutic Margins in a Novel Preclinical Model of Retinitis Pigmentosa

“…Detector sensitivity was set between 80 and 100 to avoid pixel saturation. Nine successive frames were acquired in high-speed mode (8.9 images/s) and a 55°field, and frames were aligned, averaged, and saved in non-normalized mode (Sparrow et al, 2013).…”