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      Functional Relevance and Structural Correlates of Near Infrared and Short Wavelength Fundus Autofluorescence Imaging in ABCA4-Related Retinopathy

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          Abstract

          Purpose

          To evaluate the functional relevance and structural correlates of autofluorescence (AF) alterations under short-wavelength (SW) and near-infrared (NIR) excitation light in ABCA4-related retinopathy.

          Methods

          In this prospective, cross-sectional case series, 88 eyes of 44 patients with ABCA4-related retinopathy (mean age, 37.6 years; range, 9–77 years) underwent SW-AF and NIR-AF imaging. The AF images were graded for disease characteristic patterns by two independent readers and correlated with alterations in optical coherence tomography (OCT) and impairment of retinal sensitivity along a foveo-papillary line assessed by fundus-controlled microperimetry.

          Results

          A centrifugal sequence of AF patterns from atrophic lesions to homogeneous background was found for both AF modalities. The eccentricity of each AF pattern in NIR-AF was larger compared to those in SW-AF ( P < 0.001). Increasing eccentricity of each pattern correlated with increasing retinal sensitivity. The distant border of the zone of hyperfluorescent flecks in SW-AF and hypoautofluorescent flecks in NIR-AF correlated with the margins of the ellipsoid zone loss in OCT ( r = 0.979 and r = 0.971, P < 0.001). The expansion of hypofluorescent flecks in SW-AF was associated with the boundaries of external limiting membrane loss ( r = 0.933, P < 0.001).

          Conclusions

          SW-AF and NIR-AF revealed a characteristic sequence of AF patterns that correlated with functional and structural alterations, suggesting different stages in disease progression.

          Translational Relevance

          Alterations in NIR-AF exceeded those in SW-AF images, substantiating the hypothesis of different AF origins and suggesting NIR-AF as surrogate marker for early disease-related changes.

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          Most cited references38

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          In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.

          To characterize the intrinsic fluorescence (autofluorescence) of the human ocular fundus with regard to its excitation and emission spectra, age relationship, retinal location, and topography, and to identify the dominant fluorophore among the fundus layers. Using a novel fundus spectrophotometer, fluorescence measurements were made at 7 degrees temporal to the fovea and at the fovea in 30 normal subjects and in 3 selected patients. Topographic measurements were made in 3 subjects. Ex vivo measurements of fluorescence of human retinal pigment epithelium (RPE) were obtained and compared to in vivo data. Fundus fluorescence reveals a broad band of emission between 500 and 750 nm, a maximum of approximately 630 nm, and optimal excitation of approximately 510 nm. Exhibiting a significant increase with age, this fluorescence is highest at 7 degrees to 15 degrees from the fovea, shows a well-defined foveal minimum, and decreases toward the periphery. In vivo fluorescence spectra are consistent with those obtained ex vivo on human RPE. Measurements with short wavelength excitation are strongly influenced by ocular media absorption and reveal an additional minor fluorophore in the fovea. Spectral characteristics, correlation with age, topographic distribution, and retinal location between the choriocapillaris and the photoreceptors suggest that the dominant fundus fluorophore is RPE lipofuscin. The minor fluorophore is probably in the neurosensory retina but has not been identified.
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            Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin.

            To evaluate the origin of the near-infrared autofluorescence (AF) of the fundus detected by scanning laser ophthalmoscopy and compare the distribution of this AF with that of lipofuscin. AF [787] fundus images (excitation [Exc.] 787 nm; emission [Emi.] >800 nm) were recorded with a confocal scanning laser ophthalmoscope, in 85 normal subjects (ages: 11-77 years) and in 25 patients with AMD and other retinal diseases. Standard AF [488] images (Exc. 488 nm; Emi. >500 nm) were recorded in a subset of the population. The fovea exhibits higher AF[787] than the perifovea in an area approximately 8 degrees in diameter, roughly equivalent to the area of higher RPE melanin seen in AF[488] and color images. The ratio of foveal to perifoveal AF[787] decreases with age (P < 0.0001) and is higher in subjects with light irides (P = 0.04). Higher AF[787] emanates from hyperpigmentation, from the choroidal pigment (nevi, outer layers) and from the pigment epithelium and stroma of the iris. Low AF[787] is observed in geographic atrophy particularly in subjects with light irides. AF[787] originates from the RPE and to a varying degree from the choroid. Oxidized melanin, or compounds closely associated with melanin, contributes substantially to this AF, but other fluorophores cannot be excluded at this stage. Confocal AF[787] imaging may provide a new modality to visualize pathologic features of the RPE and the choroid, and, together with AF[488] imaging, offers a new tool to study biological changes associated with aging of the RPE and pathology.
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              The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.

              To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells. Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.
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                Author and article information

                Journal
                Transl Vis Sci Technol
                Transl Vis Sci Technol
                tvst
                Transl Vis Sci Technol
                TVST
                Translational Vision Science & Technology
                The Association for Research in Vision and Ophthalmology
                2164-2591
                November 2019
                20 December 2019
                : 8
                : 6
                : 46
                Affiliations
                [1 ]University of Bonn, Department of Ophthalmology, Bonn, Germany
                [2 ]University of Bonn, Center for Rare Diseases Bonn (ZSEB), Bonn, Germany
                [3 ]Moorfields Eye Hospital NHS Foundation Trust, London, UK
                [4 ]Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
                [5 ]University of Oxford, Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, Oxford, UK
                Author notes
                Correspondence: Philipp Herrmann, Department of Ophthalmology, University of Bonn, Ernst-Abbe-Straße 2, D-53127 Bonn, Germany. e-mail: philipp.herrmann@ 123456ukbonn.de
                Article
                tvst-08-06-20 TVST-19-1694
                10.1167/tvst.8.6.46
                6927733
                31879568
                4bade390-6bcd-4e64-8981-375f7db11ba9
                Copyright 2019 The Authors

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

                History
                : 12 June 2019
                : 18 October 2019
                Categories
                Articles

                stargardt disease,retina,optical coherence tomography,microperimetry,structure–function correlation

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