Fundus Autofluorescence and RPE Lipofuscin in Age-Related Macular Degeneration

Genes that increase susceptibility to age-related macular degeneration (AMD) have been identified; however, since many individuals carrying these risk alleles do not develop disease, other contributors are involved. One additional factor, long implicated in the pathogenesis of AMD, is the lipofuscin of retinal pigment epithelium (RPE). The fluorophores that constitute RPE lipofuscin also serve as a source of autofluorescence (AF) that can be imaged by confocal laser ophthalmoscopy. The AF originating from lipofuscin is excited by the delivery of short wavelength (SW) light. A second autofluorescence is emitted from the melanin of RPE (and choroid) upon near-infrared (NIR-AF) excitation. SW-AF imaging is currently used in the clinical management of retinal disorders and the advantages of NIR-AF are increasingly recognized. Here we visit the damaging properties of RPE lipofuscin that could be significant when expressed on a background of genetic susceptibility. To advance interpretations of disease-related patterns of fundus AF in AMD, we also consider the photochemical and spectrophotometric features of the lipofuscin compounds responsible for generating the fluorescence emission

Iron overload and chelation modulates bisretinoid levels in the retina

AimIron dysregulation in conjunction with other disease processes may exacerbate retinal degeneration. We employed models of iron overload and iron chelation to explore the interactions between iron-catalyzed oxidation and photoreactive bisretinoid lipofuscin.MethodsThe mice were injected intravitreally with ferric ammonium citrate (FAC) or were treated using the iron chelator deferiprone (DFP) from birth to 2 months of age. Short-wavelength fundus autofluorescence (SW-AF) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired. The bisretinoid levels were quantified using ultra performance liquid chromatography (UPLC) and in vivo through quantitative fundus autofluorescence (qAF). In histologic sections, the photoreceptor cell viability was assessed by measuring the thickness of the outer nuclear layer (ONL).ResultsThe levels of bisretinoids, all-trans-retinal dimers, and A2PE were significantly increased in the FAC-injected eyes of C57BL/6J mice. Seven days after FAC injection, hyperautofluorescent foci were visible in fundus autofluorescence (488 nm) images, and in SD-OCT scans, aberrant hyperreflectivity was present in the outer retina and ONL thinning was observed. In FAC-injected Abca4–/– mice with pronounced RPE bisretinoid lipofuscin accumulation, the hyperautofluorescent puncta were more abundant than in the wild-type mice, and the extent of ONL thinning was greater. Conversely, the intravitreal injection of FAC in Mertk–/– mice led to a more modest increase in A2PE after 2 days. In contrast to the effect of iron accumulation, chelation with DFP resulted in significantly increased levels of A2E and A2-GPE and qAF due to the reduced iron-catalyzed oxidation of bisretinoids. In Mertk–/– mice, the A2E level was significantly lower and the ONL area was smaller than in DFP-treated mice. DFP chelation did not impair the visual cycle in BALB/cJ mice.ConclusionIron accumulation was associated with progressive impairment in photoreceptor cells that was associated with the increased formation of a bisretinoid species known to form in photoreceptor outer segments as a precursor to A2E. Additionally, disease features such as the development of hyperautofluorescence puncta in fundus AF images, hyperreflectivity in the outer retina of SD-OCT scans, and ONL thinning were more pronounced when iron was delivered to Abca4–/– mice with a greater propensity for bisretinoid formation. Higher bisretinoid levels and enhanced qAF are indicative of lesser bisretinoid loss due to oxidation

Iron promotes oxidative cell death caused by bisretinoids of retina

Intracellular Fe plays a key role in redox active energy and electron transfer. We sought to understand how Fe levels impact the retina, given that retinal pigment epithelial (RPE) cells are also challenged by accumulations of vitamin A aldehyde adducts (bisretinoid lipofuscin) that photogenerate reactive oxygen species and photodecompose into damaging aldehyde- and dicarbonyl-bearing species. In mice treated with the Fe chelator deferiprone (DFP), intracellular Fe levels, as reflected in transferrin receptor mRNA expression, were reduced. DFP-treated albino Abca4−/− and agouti wild-type mice exhibited elevated bisretinoid levels as measured by high-performance liquid chromatography or noninvasively by quantitative fundus autofluorescence. Thinning of the outer nuclear layer, a parameter indicative of the loss of photoreceptor cell viability, was also reduced in DFP-treated albino Abca4−/−. In contrast to the effects of the Fe chelator, mice burdened with increased intracellular Fe in RPE due to deficiency in the Fe export proteins hephaestin and ceruloplasmin, presented with reduced bisretinoid levels. These findings indicate that intracellular Fe promotes bisretinoid oxidation and degradation. This interpretation was supported by experiments showing that DFP decreased the oxidative/degradation of the bisretinoid A2E in the presence of light and reduced cell death in cell-based experiments. Moreover, light-independent oxidation and degradation of A2E by Fenton chemistry products were evidenced by the consumption of A2E, release of dicarbonyls, and generation of oxidized A2E species in cell-free assays

Intraretinal Correlates of Reticular Pseudodrusen Revealed by Autofluorescence and En Face OCT.

Purpose We sought to determine whether information revealed from the reflectance, autofluorescence, and absorption properties of RPE cells situated posterior to reticular pseudodrusen (RPD) could provide insight into the origins and structure of RPD. Methods RPD were studied qualitatively by near-infrared fundus autofluorescence (NIR-AF), short-wavelength fundus autofluorescence (SW-AF), and infrared reflectance (IR-R) images, and the presentation was compared to horizontal and en face spectral domain optical coherence tomographic (SD-OCT) images. Images were acquired from 23 patients (39 eyes) diagnosed with RPD (mean age 80.7 ± 7.1 [SD]; 16 female; 4 Hispanics, 19 non-Hispanic whites). Results In SW-AF, NIR-AF, and IR-R images, fundus RPD were recognized as interlacing networks of small scale variations in IR-R and fluorescence (SW-AF, NIR-AF) intensities. Darkened foci of RPD colocalized in SW-AF and NIR-AF images, and in SD-OCT images corresponded to disturbances of the interdigitation (IZ) and ellipsoid (EZ) zones and to more pronounced hyperreflective lesions traversing photoreceptor-attributable bands in SD-OCT images. Qualitative assessment of the outer nuclear layer (ONL) revealed thinning as RPD extended radially from the outer to inner retina. In en face OCT, hyperreflective areas in the EZ band correlated topographically with hyporeflective foci at the level of the RPE. Conclusions The hyperreflective lesions corresponding to RPD in SD-OCT scans are likely indicative of degenerating photoreceptor cells. The darkened foci at positions of RPD in NIR-AF and en face OCT images indicate changes in the RPE monolayer with the reduced NIR-AF and en face OCT signal suggesting a reduction in melanin that could be accounted for by RPE thinning

Hyperautofluorescent Dots are Characteristic in Ceramide Kinase Like-associated Retinal Degeneration.

There is a lack of studies which seek to discern disease expression in patients with mutations that alter retinal ceramide metabolism, specifically in the ceramide kinase like (CERKL) gene. This cross-sectional case series reports a novel phenotypic manifestation of CERKL-associated retinopathy. Four unrelated patients with homozygous CERKL mutations underwent a complete ocular exam, spectral-domain optical coherence tomography, short-wavelength fundus autofluorescence (SW-AF), quantitative autofluorescence (qAF), and full-field electroretinogram (ffERG). Decreased visual acuity and early-onset maculopathy were present in all patients. All four patients had extensive hyperautofluorescent foci surrounding an area of central atrophy on SW-AF imaging, which has not been previously characterized. An abnormal spatial distribution of qAF signal was seen in one patient, and abnormally elevated qA

Quantitative Fundus Autofluorescence and Optical Coherence Tomography in ABCA4 Carriers

PURPOSE. To assess whether carriers of ABCA4 mutations have increased RPE lipofuscin levels based on quantitative fundus autofluorescence (qAF) and whether spectral-domain optical coherence tomography (SD-OCT) reveals structural abnormalities in this cohort. METHODS. Seventy-five individuals who are heterozygous for ABCA4 mutations (mean age, 47.3 years; range, 9-82 years) were recruited as family members of affected patients from 46 unrelated families. For comparison, 57 affected family members with biallelic ABCA4 mutations (mean age, 23.4 years; range, 6-67 years) and two noncarrier siblings were also enrolled. Autofluorescence images (308, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference. The gray levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density to yield qAF. Horizontal SD-OCT scans through the fovea were obtained and the thicknesses of the outer retinal layers were measured. RESULTS. In 60 of 65 carriers of ABCA4 mutations (age range, 9-60), qAF levels were within normal limits (95% confidence level) observed for healthy noncarrier subjects, while qAF levels of affected family members were significantly increased. Perifoveal fleck-like abnormalities were observed in fundus AF images in four carriers, and corresponding changes were detected in the outer retinal layers in SD-OCT scans. Thicknesses of the outer retinal layers were within the normal range. CONCLUSIONS. With few exceptions, individuals heterozygous for ABCA4 mutations and between the ages of 9 and 60 years do not present with elevated qAF. In a small number of carriers, perifoveal fleck-like changes were visible

Patterns and Intensities of Near-Infrared and Short-Wavelength Fundus Autofluorescence in Choroideremia Probands and Carriers

PURPOSE. To ascertain cellular constituents within islands of preserved retina in choroideremia (CHM) by multimodal imaging. METHODS. CHM probands (16) and female carriers (9) of CHM were studied. Near-infrared autofluorescence (NIR-AF; 787-nm excitation; emission, > 830 nm), short-wavelength autofluorescence (SW-AF; 488-nm excitation, 500-to 680-nm emission), and spectral-domain optical coherence tomography (SD-OCT) images were acquired with a confocal scanning laser ophthalmoscope. SW-AF intensities were measured by quantitative fundus autofluorescence (qAF), and NIR-AF intensity profiles were analyzed. Retinal thicknesses and visual acuity were measured. RESULTS. In 19 of 31 eyes of affected males, islands of preserved NIR-AF signal were also visible as fluorescence signal in SW-AF images. Notable in 12 eyes were areas of speckled SW-AF that was hypoautofluorescent in the NIR-AF image. Islands of preserved NIR-AF and SW-AF signal were often associated with the presence of visible but thinned outer nuclear layer and discontinuous interdigitation zone, ellipsoid zone, and external limiting membrane. NIR-AF profiles revealed that even in areas of preserved retina, the NIR-AF signal from retinal pigment epithelium (RPE) melanin is greatly reduced. qAF was reduced overall. The fundus of carriers was characterized by a mosaicism in which patches of reduced NIR-AF colocalized with reduced SW-AF. CONCLUSIONS. In CHM-affected males, the presence of RPE was indicated by an NIR-AF signal and the absence of hypertransmission of OCT signal into the choroid. RPE preservation was associated with better visual acuity. In carriers, patches of reduced SW-AF colocalized with decreased NIR-AF and qAF was severely reduced.Peer reviewe

Electroretinography Reveals Difference in Cone Function between Syndromic and Nonsyndromic USH2A Patients

Usher syndrome is an inherited and irreversible disease that manifests as retinitis pigmentosa (RP) and bilateral neurosensory hearing loss. Mutations in Usherin 2A (USH2A) are not only a frequent cause of Usher syndrome, but also nonsyndromic RP. Although gene-and cell-based therapies are on the horizon for RP and Usher syndrome, studies characterizing natural disease are lacking. In this retrospective analysis, retinal function of USH2A patients was quantified with electroretinography. Both groups had markedly reduced rod and cone responses, but nonsyndromic USH2A patients had 30 Hz-flicker electroretinogram amplitudes that were significantly higher than syndromic patients, suggesting superior residual cone function. There was a tendency for Usher syndrome patients to have a higher distribution of severe mutations, and alleles in this group had a higher odds of containing nonsense or frame-shift mutations. These data suggest that the previously reported severe visual phenotype seen in syndromic USH2A patients could relate to a greater extent of cone dysfunction. Additionally, a genetic threshold may exist where mutation burden relates to visual phenotype and the presence of hearing deficits. The auditory phenotype and allelic hierarchy observed among patients should be considered in prospective studies of disease progression and during enrollment for future clinical trials.National Institute of HealthNational Cancer InstituteResearch to Prevent Blindness (RPB)RPB, New York, NY, USARPBInternational Council of Ophthalmology - Retina Research FoundationNIHTistou and Charlotte Kerstan FoundationSchneeweiss Stem Cell Fund, New York StateFoundation Fighting Blindness New York Regional Research CenterCrowley Family FundGebroe Family FoundationColumbia Univ, Dept Ophthalmol, Med Ctr, Jonas Childrens Vis Care, New York, NY 10027 USAColumbia Univ, Dept Ophthalmol, Med Ctr, Bernard & Shirlee Brown Glaucoma Lab, New York, NY 10027 USANew York Presbyterian Hosp, Edward S Harkness Eye Inst, New York, NY 10034 USASuny Downstate Med Ctr, Brooklyn, NY 11203 USAUniv Fed Espirito Santo, Dept Ophthalmol, Vitoria, BrazilUniv Fed Sao Paulo, Dept Ophthalmol, Sao Paulo, BrazilColumbia Univ, Dept Biostat, New York, NY USAUniv Montreal, Dept Ophthalmol, Montreal, PQ, CanadaShanghai Jiao Tong Univ, Sch Med, Xin Hua Hosp, Dept Ophthalmol, Shanghai, Peoples R ChinaColumbia Univ, Inst Human Nutr, Dept Pathol & Cell Biol, Stem Cell Initiat CSCI,Coll Phys & Surg, New York, NY 10032 USAStanford Univ, Dept Ophthalmol, Om Lab, Byers Eye Inst, Palo Alto, CA 94304 USAUniv Fed Sao Paulo, Dept Ophthalmol, Sao Paulo, BrazilWeb of Scienc