The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL-4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.

Purpose To investigate the incidence of reticular macular disease (RMD), a subphenotype of age-related macular degeneration (AMD), in multilobular geographic atrophy (GA) and its relation to GA progression. Methods 157 eyes of 99 subjects with AMD, primary GA, and good-quality autofluorescence (AF) and/or infrared (IR) images were classified into unilobular GA (1 lesion) or multilobular GA (>= 2 distinct and/or coalescent lesions). 34 subjects (50 eyes) had serial imaging. We determined the spatiotemporal relationships of RMD to GA and GA progression rates in 5 macular fields. Results 144/157 (91.7%) eyes had multilobular GA, 95.8% of which exhibited RMD. In subjects with serial imaging, the mean GA growth rate significantly differed between the unilobular and multilobular groups (0.40 mm2 /yrvs. 1.30 mm2 /yr, p< 0.001). Of the macular fields in these eyes, 77.1% of fields with RMD at baseline showed subsequent GA progression, while 53.4% of fields without RMD showed progression (p <0.001). Percentage of fields with RMD significantly correlated with GA progression rate(p=0.01). Conclusion AF and IR imaging demonstrates that RMD is nearly always present with multilobular GA in AMD. Further, GA lobules frequently develop in areas of RMD, suggesting progression of a single underlying disease process.

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...

Haploinsufficiency of TBX1, encoding a T-box transcription factor, is largely responsible for the physical malformations in velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2, could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right-sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype enrichment groups. Nine common SNPs (MAF >0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome.

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.