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... One study of five participants by Teussink et al. 122 assessed the effects of light exposure protection using a black contact lens covering > 90% of light in the visible spectrum, worn on the better eye during waking hours for a year. 122 The other eye acted as a control. ...
... One study of five participants by Teussink et al. 122 assessed the effects of light exposure protection using a black contact lens covering > 90% of light in the visible spectrum, worn on the better eye during waking hours for a year. 122 The other eye acted as a control. The study was undertaken in the Netherlands. ...
... There was some controversy over the role of genetic testing, with a theory put forward by Awh and colleagues 350 that only some genetic subgroups benefited from antioxidants and zinc, and that some were actually harmed. This theory was refuted by Chew et al. 122 However, there may be different progression rates according to genetic factors. Seddon et al. 351 reported higher rates of progression in genotypes CFH Y402H (CC) and ARMS2 (TT). ...
Article
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Background Age-related macular degeneration (AMD) is the leading cause of visual loss in older people. Advanced AMD takes two forms, neovascular (wet) and atrophic (dry). Stargardt disease (STGD) is the commonest form of inherited macular dystrophy. Objective To carry out a systematic review of treatments for dry AMD and STGD, and to identify emerging treatments where future NIHR research might be commissioned. Design Systematic review. Methods We searched MEDLINE, EMBASE, Web of Science and The Cochrane Library from 2005 to 13 July 2017 for reviews, journal articles and meeting abstracts. We looked for studies of interventions that aim to preserve or restore vision in people with dry AMD or STGD. The most important outcomes are those that matter to patients: visual acuity (VA), contrast sensitivity, reading speed, ability to drive, adverse effects of treatment, quality of life, progression of disease and patient preference. However, visual loss is a late event and intermediate predictors of future decline were accepted if there was good evidence that they are strong predictors of subsequent visual outcomes. These include changes detectable by investigation, but not necessarily noticed by people with AMD or STGD. ClinicalTrials.gov, the World Health Organization search portal and the UK Clinical Trials gateway were searched for ongoing and recently completed clinical trials. Results The titles and abstracts of 7948 articles were screened for inclusion. The full text of 398 articles were obtained for further screening and checking of references and 112 articles were included in the final report. Overall, there were disappointingly few good-quality studies (including of sufficient size and duration) reporting useful outcomes, particularly in STGD. However we did identify a number of promising research topics, including drug treatments, stem cells, new forms of laser treatment, and implantable intraocular lens telescopes. In many cases, research is already under way, funded by industry or governments. Limitations In AMD, the main limitation came from the poor quality of much of the evidence. Many studies used VA as their main outcome despite not having sufficient duration to observe changes. The evidence on treatments for STGD is sparse. Most studies tested interventions with no comparison group, were far too short term, and the quality of some studies was poor. Future work We think that the topics on which the Health Technology Assessment (HTA) and Efficacy Mechanism and Evaluation (EME) programmes might consider commissioning primary research are in STGD, a HTA trial of fenretinide (ReVision Therapeutics, San Diego, CA, USA), a visual cycle inhibitor, and EME research into the value of lutein and zeaxanthin supplements, using short-term measures of retinal function. In AMD, we suggest trials of fenretinide and of a potent statin. There is epidemiological evidence from the USA that the drug, levodopa, used for treating Parkinson’s disease, may reduce the incidence of AMD. We suggest that similar research should be carried out using the large general practice databases in the UK. Ideally, future research should be at earlier stages in both diseases, before vision is impaired, using sensitive measures of macular function. This may require early detection of AMD by screening. Study registration This study is registered as PROSPERO CRD42016038708. Funding The National Institute for Health Research HTA programme.
... 51 The photo-oxidative processes initiated by RPE bisretinoid could explain suggested links to light exposure in both STGD1 and AMD. [52][53][54][55] In a study of fundus flecks in STGD1, it was found that the NIR-AF signal generated largely by RPE melanin is absent at positions of flecks visible in photoreceptor-attributable bands of SD-OCT images. Thus, Sparrow suggested that the shortwavelength autofluorescence signal of flecks originates in the augmented bisretinoid of impaired photoreceptor cells. ...
Article
Accumulation of fluorescent metabolic byproducts of the visual (retinoid) cycle is associated with photoreceptor and retinal pigment epithelial cell death in both Stargardt disease and atrophic (nonneovascular) age-related macular degeneration (AMD). As a consequence of this observation, small molecular inhibitors of enzymes in the visual cycle were recently tested in clinical trials as a strategy to protect the retina and retinal pigment epithelium in patients with atrophic AMD. To address the clinical translational needs for therapies aimed at both diseases, a workshop organized by the Foundation Fighting Blindness was hosted by the Department of Pharmacology at Case Western Reserve University on February 17, 2017, at the Tinkham Veale University Center, Cleveland, OH, USA. Invited speakers highlighted recent advances in the understanding of the pathophysiology of Stargardt disease, in terms of its clinical characterization and the development of endpoints for clinical trials, and discussed the comparability of therapeutic strategies between atrophic age-related macular degeneration (AMD) and Stargardt disease. Investigators speculated that reducing the concentrations of visual cycle precursor substances and/or their byproducts may provide valid therapeutic options for the treatment of Stargardt disease. Here we review the workshop's presentations in the context of published literature to help shape the aims of ongoing research endeavors and aid the development of therapies for Stargardt disease.
... There are several pieces of evidence suggesting that the decrease in fluorescence in the retina could be due, at least in part, to photobleaching. In four out of five Stargardt disease patients monitored over a period of one year or longer, an overall reduction in lipofuscin fluorescence induced by excitation with 488 nm light intensity was reported, despite no cell loss occurring [21]. In this study, the reduction in fluorescence intensity was smaller in fellow eyes where patients' corneas were covered during the daytime by contact lenses blocking above 90% of the visible light entering the eye. ...
Article
Full-text available
Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes.
... There are several pieces of evidence suggesting that the decrease in fluorescence in the retina could be due, at least in part, to photobleaching. In four out of five Stargardt disease patients monitored over a period of one year or longer, an overall reduction in lipofuscin fluorescence induced by excitation with 488 nm light intensity was reported, despite no cell loss occurring [21]. In this study, the reduction in fluorescence intensity was smaller in fellow eyes where patients' corneas were covered during the daytime by contact lenses blocking above 90% of the visible light entering the eye. ...
Preprint
Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE) where its fluorescence properties are used to assess the retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE, and lipofuscin-laden-cells to visible light, and determine whether an abundant component of lipofuscin, docosahexaenoate (DHA) can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible leads to a decrease of its long-wavelength fluorescence at about 610 nm with concomitant growth of the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure to light of lipofuscin-laden cells leads to loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes of fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra together with quantitation of intensity of long-wavelength fluorescence can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, thereby useful for screening the retina for increased oxidative damage and early AMD-related changes.
... One study of Stargardt patients reported that unilateral protection from light exposure led to reduced progression of maculopathy, suggesting a reduction in the rate of RPE damage in the protected eye. [9] We propose that the aphakic status of the left eye in our patient may have been protective by leading to a decrease in light damage in that eye. Two potential mechanisms are as follows: (1) lack of focused light in the aphakic left eye when not wearing contact lens correction and (2) an alteration of the light spectrum reaching the retina due to the properties of the rigid contact lens. ...
... An indispensable part of NGS data analysis is access to genome sequencing data with a large number of samples, which provides a basic tool in determining pathogenicity of ABCA4 variants. Identification of causal ABCA4 mutations is of practical relevance for affected families as precautions that may reduce disease progression, such as protection from excessive light exposure and avoidance of vitamin A supplementation, should be followed until therapy options become available (Haji Abdollahi and Hirose, 2013;Paskowitz et al., 2006;Radu et al., 2008;Teussink et al., 2015;Weng et al., 1999). ...
Article
Variation in the ABCA4 locus has emerged as the most prevalent cause of monogenic retinal diseases. The study aimed to discover causative ABCA4 mutations in a large but not previously investigated cohort with ABCA4-related diseases originating from Central Europe and to refine the genetic relevance of all identified variants based on population evidence. Comprehensive clinical studies were performed to identify patients with Stargardt disease (STGD, n=76) and cone-rod dystrophy (CRD, n=16). Next-generation sequencing targeting ABCA4 was applied for a widespread screening of the gene. The results were analyzed in the context of exome data from a corresponding population (n=594) and other large genomic databases. Our data disprove the pathogenic status of p.V552I and provide more evidence against a causal role of four further ABCA4 variants as drivers of the phenotype under a recessive paradigm. The study identifies 12 novel potentially pathogenic mutations (four of them recurrent) and a novel complex allele p.[(R152*; V2050L)]. In one third (31/92) of our cohort we detected the p.[(L541P; A1038V)] complex allele, which represents an unusually high level of genetic homogeneity for ABCA4-related diseases. Causative ABCA4 mutations account for 79% of STGD and 31% of CRD cases. A combination of p.[(L541P; A1038V)] and/or a truncating ABCA4 mutation always resulted in an early disease onset. Identification of ABCA4 retinopathies provides a specific molecular diagnosis and justifies a prompt introduction of simple precautions that may slow disease progression. The comprehensive, population-specific study expands our knowledge on the genetic landscape of retinal diseases.
... Patients are currently advised to avoid supplements containing vitamin A, due to lipofuscin accumulation being seen in Abca4 knockout mice that were given vitamin A [163]. Wearing protective, dark-tinted glasses in bright conditions is recommended to reduce short wavelength light reaching the retina, thus reducing the risk of light toxicity [164]. Potential treatments currently being investigated include pharmacological interventions, gene therapy, and stem cell-based therapy approaches (see Table 6). ...
Article
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Stargardt disease (STGD1) and ABCA4 retinopathies (ABCA4R) are caused by pathogenic variants in the ABCA4 gene inherited in an autosomal recessive manner. The gene encodes an importer flippase protein that prevents the build-up of vitamin A derivatives that are toxic to the RPE. Diagnosing ABCA4R is complex due to its phenotypic variability and the presence of other inherited retinal dystrophy phenocopies. ABCA4 is a large gene, comprising 50 exons; to date >2000 variants have been described. These include missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of variants in ABCA4. However, in a significant proportion of patients with an ABCA4R phenotype, a second variant in ABCA4 is not identified. This could be due to the presence of yet unknown variants, or hypomorphic alleles being incorrectly classified as benign, or the possibility that the disease is caused by a variant in another gene. This underlines the importance of accurate genetic testing. The pathogenicity of novel variants can be predicted using in silico programs, but these rely on databases that are not ethnically diverse, thus highlighting the need for studies in differing populations. Functional studies in vitro are useful towards assessing protein function but do not directly measure the flippase activity. Obtaining an accurate molecular diagnosis is becoming increasingly more important as targeted therapeutic options become available; these include pharmacological, gene-based, and cell replacement-based therapies. The aim of this review is to provide an update on the current status of genotyping in ABCA4 and the status of the therapeutic approaches being investigated.
... For instance, sunglasses that attenuate over a broad range of wavelengths or yellow lenses that reduce blue wavelengths could be expected to provide protection (71). A black contact lens that blocked >90% of light in one eye of STGD1 patients was found to reduce the progression of decreased fundus AF (72); the latter decrease is likely attributable to bisretinoid photooxidation and photodegradation. ...
Article
Although currently available treatment options for age-related macular degeneration (AMD) are limited, particularly for atrophic AMD, the identification of predisposing genetic variations has informed clinical studies addressing therapeutic options such as complement inhibitors and anti-inflammatory agents. To lower risk of early AMD, recommended lifestyle interventions such as the avoidance of smoking and the intake of low glycemic antioxidant-rich diets have largely followed from the identification of nongenetic modifiable factors. On the other hand, the challenge of understanding the complex relationship between aging and cumulative damage leading to AMD has fueled investigations of the visual cycle adducts that accumulate in retinal pigment epithelial (RPE) cells and are a hallmark of aging retina. These studies have revealed properties of these compounds that provide insights into processes that may compromise RPE and could contribute to disease mechanisms in AMD. This work has also led to the design of targeted therapeutics that are currently under investigation.
... Because the photodegradative processes examined here could over time be responsible for cumulative damage to retina and Bruch's membrane, the present findings have implications with respect to development of therapeutics that address bisretinoid lipofuscin deposition in recessive Stargardt disease and AMD (55-60) and justify efforts to protect the retina of STGD1 patients by reducing light exposure through the use of a black contact lens (61). Quantitative Fundus Autofluorescence. ...
Article
Adducts of retinaldehyde (bisretinoids) form nonenzymatically in photoreceptor cells and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin; these fluorophores are implicated in the pathogenesis of inherited and age-related macular degeneration (AMD). Here we demonstrate that bisretinoid photodegradation is ongoing in the eye. High-performance liquid chromatography (HPLC) analysis of eyes of dark-reared and cyclic light-reared wild-type mice, together with comparisons of pigmented versus albino mice, revealed a relationship between intraocular light and reduced levels of the bisretinoids A2E and A2-glycero-phosphoethanolamine (A2-GPE). Analysis of the bisretinoids A2E, A2-GPE, A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), and all-trans-retinal dimer-phosphatidylethanolamine (all-trans-retinal dimer-PE) also decreases in albino Abca4(-/-) mice reared in cyclic light compared with darkness. In albino Abca4(-/-) mice receiving a diet supplemented with the antioxidant vitamin E, higher levels of RPE bisretinoid were evidenced by HPLC analysis and quantitation of fundus autofluorescence; this effect is consistent with photooxidative processes known to precede bisretinoid degradation. Amelioration of outer nuclear layer thinning indicated that vitamin E treatment protected photoreceptor cells. Conversely, in-cage exposure to short-wavelength light resulted in reduced fundus autofluorescence, decreased HPLC-quantified A2E, outer nuclear layer thinning, and increased methylglyoxal (MG)-adducted protein. MG was also released upon bisretinoid photodegradation in cells. We suggest that the lower levels of these diretinal adducts in cyclic light-reared and albino mice reflect photodegradative loss of bisretinoid. These mechanisms may underlie associations among AMD risk, oxidative mechanisms, and lifetime light exposure.
... Interestingly, in a light exposure study, five patients with STGD1 who wore a black contact lens in one eye during waking hours for 12 months were observed to have less progression on FAF imaging in their study eye compared with the fellow eye. 72 Avenues of intervention STGD1 is currently subject to more clinical trials than any other inherited retinal disease, with gene replacement, stem cell therapy and pharmacological approaches. ...
Article
Full-text available
Stargardt disease (STGD1; MIM 248200) is the most prevalent inherited macular dystrophy and is associated with disease-causing sequence variants in the gene ABCA4. Significant advances have been made over the last 10 years in our understanding of both the clinical and molecular features of STGD1, and also the underlying pathophysiology, which has culminated in ongoing and planned human clinical trials of novel therapies. The aims of this review are to describe the detailed phenotypic and genotypic characteristics of the disease, conventional and novel imaging findings, current knowledge of animal models and pathogenesis, and the multiple avenues of intervention being explored.
... Whether the mechanism of photochemical damage involves changes in either lipofuscin or molecules within the visual cycle such as all-trans-retinal, patients with STGD1 will be highly susceptible to photic injury [14,16]. Consistent with this notion, even chronic exposure to normal daylight appears to increase the progression of RPE damage in STGD1 [19]. Evidence from studies with Abca4 -/mice indicates that an accelerated accumulation of lipofuscin bisretinoids in the RPE mainly underlies increases in photosensitivity in STGD1 [18]. ...
Article
Full-text available
Purpose Current standards and guidelines aimed at preventing retinal phototoxicity during intentional exposures do not specifically evaluate the contribution of endogenous photosensitizers. However, certain retinal diseases are characterized by abnormal accumulations of potential photosensitizers such as lipofuscin bisretinoids in the retinal pigment epithelium (RPE). We sought to determine these contributions by a numerical assessment of in-vivo photo-oxidative stress during irradiation of RPE lipofuscin. Methods Based on the literature, we calculated the retinal exposure levels, optical filtering of incident radiation by the ocular lens, media, photoreceptors, and RPE melanin, light absorption by lipofuscin, and photochemical effects in the RPE in two situations: exposure to short-wavelength (λ = 488 nm) fundus autofluorescence (SW-AF) excitation light and exposure to indirect (diffuse) sunlight. Results In healthy persons at age 20, 40, and 60, respectively, the rate of oxygen photoconsumption by lipofuscin increases by 1.3, 1.7, and 2.4 fold during SW-AF-imaging as compared to diffuse sunlight. In patients with STGD1 below the age of 30, this rate was 3.3-fold higher compared to age-matched controls during either sunlight or SW-AF imaging. Conclusions Our results suggest that the RPE of patients with STGD1 is generally at increased risk of photo-oxidative stress, while exposure during SW-AF-imaging amplifies this risk. These theoretical results have not yet been verified with in-vivo data due to a lack of sufficiently sensitive in-vivo measurement techniques.
... Flecks are generally regarded as a biomarker of disease severity based on their emergence along the central and peripheral axis of the ABCA4-associated retinopathy retina (Cukras et al., 2012). Teussink et al. sought to study the effect of light on the progression of ABCA4associated retinopathy by comparing fleck accumulation in an eye of patients compared to the fellow eye that was continuously patched over one year, but the results were variable (Teussink et al., 2015). The causal role of flecks in the pathophysiology of ABCA4-associated retinopathy is thus far uncertain and much remains to be elucidated. ...
Article
Full-text available
The ABCA4 protein (then called a “rim protein”) was first identified in 1978 in the rims and incisures of rod photoreceptors. The corresponding gene, ABCA4, was cloned in 1997, and variants were identified as the cause of autosomal recessive Stargardt disease (STGD1). Over the next two decades, variation in ABCA4 has been attributed to phenotypes other than the classically defined STGD1 or fundus flavimaculatus, ranging from early onset and fast progressing cone-rod dystrophy and retinitis pigmentosa-like phenotypes to very late onset cases of mostly mild disease sometimes resembling, and confused with, age-related macular degeneration. Similarly, analysis of the ABCA4 locus uncovered a trove of genetic information, including >1200 disease-causing mutations of varying severity, and of all types – missense, nonsense, small deletions/insertions, and splicing affecting variants, of which many are located deep-intronic. Altogether, this has greatly expanded our understanding of complexity not only of the diseases caused by ABCA4 mutations, but of all Mendelian diseases in general. This review provides an in depth assessment of the cumulative knowledge of ABCA4-associated retinopathy – clinical manifestations, genetic complexity, pathophysiology as well as current and proposed therapeutic approaches.
... We only included patients with a reported disease onset <12 years of age [4,5]. We excluded (1) patients with very early disease in which only thickening of the external limiting membrane was present, because this would preclude the OCT measurements, (2) patients with advanced disease characterized by RPE atrophy beyond the vascular arcades at first presentation, and (3) patients who participated in an interventional trial [40]. This study was approved by the local ethics committee on Research Involving Human Subjects of the Radboud university medical center "Commissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen" and the National Research Ethics Service (NRES) Committee London-Camden & Islington, and was performed in accordance with the Declaration of Helsinki. ...
Article
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Background: Each inherited retinal disorder is rare, but together, they affect millions of people worldwide. No treatment is currently available for these blinding diseases, but promising new options-including gene therapy-are emerging. Arguably, the most prevalent retinal dystrophy is Stargardt disease. In each case, the specific combination of ABCA4 variants (> 900 identified to date) and modifying factors is virtually unique. It accounts for the vast phenotypic heterogeneity including variable rates of functional and structural progression, thereby potentially limiting the ability of phase I/II clinical trials to assess efficacy of novel therapies with few patients. To accommodate this problem, we developed and validated a sensitive and reliable composite clinical trial endpoint for disease progression based on structural measurements of retinal degeneration. Methods and findings: We used longitudinal data from early-onset Stargardt patients from the Netherlands (development cohort, n = 14) and the United Kingdom (external validation cohort, n = 18). The composite endpoint was derived from best-corrected visual acuity, fundus autofluorescence, and spectral-domain optical coherence tomography. Weighting optimization techniques excluded visual acuity from the composite endpoint. After optimization, the endpoint outperformed each univariable outcome, and showed an average progression of 0.41° retinal eccentricity per year (95% confidence interval, 0.30-0.52). Comparing with actual longitudinal values, the model accurately predicted progression (R2, 0.904). These properties were largely preserved in the validation cohort (0.43°/year [0.33-0.53]; prediction: R2, 0.872). We subsequently ran a two-year trial simulation with the composite endpoint, which detected a 25% decrease in disease progression with 80% statistical power using only 14 patients. Conclusions: These results suggest that a multimodal endpoint, reflecting structural macular changes, provides a sensitive measurement of disease progression in Stargardt disease. It can be very useful in the evaluation of novel therapeutic modalities in rare disorders.
... Full-field electroretinography (ffERG) results can be used to characterize peripheral function, while multifocal electroretinography (mfERG) results can be used to characterize central function. [39][40][41] Several interventions for STGD have been proposed, including light deprivation, 42 gene replacement therapy, 44 and human stem cell transplantation. 44,47 Information about current clinical treatment trials can be accessed at ClinicalTrials.gov. ...
Article
In this paper we review three common juvenile macular degenerations: Stargardt disease, X-linked retinoschisis, and Best vitelliform macular dystrophy. These are inherited disorders that typically present during childhood, when vision is still developing. They are sufficiently common that they should be included in the differential diagnosis of visual loss in pediatric patients. Diagnosis is secured by a combination of clinical findings, optical coherence tomography (OCT) imaging, and genetic testing. Early diagnosis promotes optimal management. While there is currently no definitive cure for these conditions, therapeutic modalities under investigation include pharmacologic treatment, gene therapy, and stem cell transplantation.
... The findings discussed here indicate that although light deprivation does not prevent the formation of bisretinoids (Boyer et al. 2012;Ueda et al. 2016), limiting light exposure can protect against damaging bisretinoid photodegradation. Thus not surprisingly, a black contact lens that blocked >90% of light in one eye of STGD1 patients was found to reduce the progression of decreased fundus autofluorescence (four of five patients) as compared with the patients' unprotected eyes (Teussink et al. 2015). Sunglasses that attenuate light over a broad range of wavelengths or yellow lenses that reduce "blue" wavelengths might also be used to advantage. ...
Chapter
Full-text available
Retinaldehyde adducts (bisretinoids) accumulate in retinal pigment epithelial (RPE) cells as lipofuscin. Bisretinoids are implicated in some inherited and age-related forms of macular degeneration that lead to the death of RPE cells and diminished vision. By comparing albino and black-eyed mice and by rearing mice in darkness and in cyclic light, evidence indicates that bisretinoid fluorophores undergo photodegradation in the eye (Ueda et al. Proc Natl Acad Sci 113:6904-6909, 2016). Given that the photodegradation products modify and impair cellular and extracellular molecules, these processes likely impart cumulative damage to retina.
... The findings discussed here indicate that although light deprivation does not prevent the formation of bisretinoids (Boyer et al. 2012;Ueda et al. 2016), limiting light exposure can protect against damaging bisretinoid photodegradation. Thus not surprisingly, a black contact lens that blocked >90% of light in one eye of STGD1 patients was found to reduce the progression of decreased fundus autofluorescence (four of five patients) as compared with the patients' unprotected eyes (Teussink et al. 2015). Sunglasses that attenuate light over a broad range of wavelengths or yellow lenses that reduce "blue" wavelengths might also be used to advantage. ...
Chapter
Full-text available
Purpose: To identify the molecular basis of inherited retinal degeneration (IRD) in a familial case of Pakistani origin using whole-exome sequencing. Methods: A thorough ophthalmic examination was completed, and genomic DNA was extracted using standard protocols. Whole exome(s) were captured with Agilent V5 + UTRs probes and sequenced on Illumina HiSeq genome analyzer. The exomeSuite software was used to filter variants, and the candidate causal variants were prioritized, examining their allele frequency and PolyPhen2, SIFT, and MutationTaster predictions. Sanger dideoxy sequencing was performed to confirm the segregation with disease phenotype and absence in ethnicity-matched control chromosomes. Results: Ophthalmic examination confirmed retinal degeneration in all affected individuals that segregated as an autosomal recessive trait in the family. Whole-exome sequencing identified two homozygous missense variants: c.1304G > A; p.Arg435Gln in ZNF408 (NM_024741) and c.902G > A; p.Gly301Asp in C1QTNF4 (NM_031909). Both variants segregated with the retinal phenotype in the PKRD320 and were absent in ethnically matched control chromosomes. Conclusion: Whole-exome sequencing coupled with bioinformatics analysis identified potential novel variants that might be responsible for IRD.
Article
Importance: Sensitive outcome measures for disease progression are needed for treatment trials of Stargardt disease. Objective: To describe the yearly progression rate of atrophic lesions in the retrospective Progression of Stargardt Disease study. Design, setting, and participants: A multicenter retrospective cohort study was conducted at tertiary referral centers in the United States and Europe. A total of 251 patients aged 6 years or older at baseline, harboring disease-causing variants in ABCA4 (OMIM 601691), enrolled in the study from 9 centers between August 2, 2013, and December 12, 2014; of these patients, 215 had at least 2 gradable fundus autofluorescence images with atrophic lesion(s) present in at least 1 eye. Exposures: Areas of definitely decreased autofluorescence (DDAF) and questionably decreased autofluorescence were quantified by a reading center. Progression rates were estimated from linear mixed models with time as the independent variable. Main outcomes and measures: Yearly rate of progression using the growth of atrophic lesions measured by fundus autofluorescence. Results: A total of 251 participants (458 study eyes) were enrolled. Images from 386 eyes of 215 participants (126 females and 89 males; mean [SD] age, 29.9 [14.7] years; mean [SD] age of onset of symptoms, 21.9 [13.3] years) showed atrophic lesions present on at least 2 visits and were graded for 2 (156 eyes), 3 (174 eyes), or 4 (57 eyes) visits. A subset of 224 eyes (123 female participants and 101 male participants; mean [SD] age, 33.0 [15.1] years) had areas of DDAF present on at least 2 visits; these eyes were included in the estimation of the progression of the area of DDAF. At the first visit, DDAF was present in 224 eyes (58.0%), with a mean (SD) lesion size of 2.2 (2.7) mm2. The total mean (SD) area of decreased autofluorescence (DDAF and questionably decreased autofluorescence) at first visit was 2.6 (2.8) mm2. Mean progression of DDAF was 0.51 mm2/y (95% CI, 0.42-0.61 mm2/y), and of total decreased fundus autofluorescence was 0.35 mm2/y (95% CI, 0.28-0.43 mm2/y). Rates of progression depended on the initial size of the lesion. Conclusions and relevance: In Stargardt disease with DDAF lesions, fundus autofluorescence may serve as a monitoring tool for interventional clinical trials that aim to slow disease progression. Rates of progression depended mainly on initial lesion size.
Chapter
Inherited retinal diseases are a group of clinically and genetically heterogeneous conditions, which can result in diagnostic challenges, thereby necessitating detailed multimodal retinal imaging, as well as electrophysiological and psychophysical evaluation. Herein, the inherited retinal diseases that primarily affect cone photoreceptors will be presented on the basis of disease natural history (stationary or progressive). Cone and cone-rod dystrophies are by definition progressive conditions and cone dysfunction syndromes are stationary. An overview of each category is followed by a more detailed review of individual conditions with consideration of potential therapeutic strategies when possible.
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Purpose: To investigate intersibling phenotypic concordance in Stargardt disease (STGD1). Design: Retrospective cohort study. Participants: Siblings with genetically confirmed STGD1 and at least 1 available fundus autofluorescence (FAF) image of both eyes. Methods: We compared age at onset within families. Disease duration was matched to investigate differences in best-corrected visual acuity (BCVA) and compared the survival time for reaching severe visual impairment (<20/200 Snellen or >1.0 logarithm of the minimum angle of resolution [logMAR]). Central retinal atrophy area was quantified independently by 2 experienced graders using semiautomated software and compared between siblings. Both graders performed qualitative assessment of FAF and spectral-domain (SD) OCT images to identify phenotypic differences. Main outcome measures: Differences in age at onset, disease duration-matched BCVA, time to severe visual impairment development, FAF atrophy area, FAF patterns, and genotypes. Results: Substantial differences in age at onset were present in 5 of 17 families, ranging from 13 to 39 years. Median BCVA at baseline was 0.60 logMAR (range, -0.20 to 2.30 logMAR; Snellen equivalent, 20/80 [range, 20/12-hand movements]) in the right eye and 0.50 logMAR (range, -0.20 to 2.30 logMAR; Snellen equivalent, 20/63 [range, 20/12-hand movements]) in the left eye. Disease duration-matched BCVA was investigated in 12 of 17 families, and the median difference was 0.41 logMAR (range, 0.00-1.10 logMAR) for the right eye and 0.41 logMAR (range, 0.00-1.08 logMAR) for the left eye. We observed notable differences in time to severe visual impairment development in 7 families, ranging from 1 to 29 years. Median central retinal atrophy area was 11.38 mm2 in the right eye (range, 1.98-44.78 mm2) and 10.59 mm2 in the left eye (range, 1.61-40.59 mm2) and highly comparable between siblings. Similarly, qualitative FAF and SD OCT phenotypes were highly comparable between siblings. Conclusions: Phenotypic discordance between siblings with STGD1 carrying the same ABCA4 variants is a prevalent phenomenon. Although the FAF phenotypes are highly comparable between siblings, functional outcomes differ substantially. This complicates both sibling-based prognosis and genotype-phenotype correlations and has important implications for patient care and management.
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Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of disorders characterised by photoreceptor degeneration or dysfunction. These disorders typically present with severe vision loss which can be progressive, with disease onset ranging from congenital to late adulthood. The advances in genetics, retinal imaging and molecular biology, have conspired to create the ideal environment for establishing treatments for IRD, with the first approved gene therapy and the commencement of multiple clinical trials. The scope of this review is to familiarize clinicians and scientists with the current management and the prospects for novel therapies for: (i) macular dystrophies, (ii) cone and cone‐rod dystrophies, (iii) cone dysfunction syndromes, (iv) Leber congenital amaurosis, (v) rod‐cone dystrophies, (vi) rod dysfunction syndromes, and (vii) chorioretinal dystrophies. We also briefly summarize the investigated end‐points for the on‐going trials.
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Age-related macular degeneration (AMD) is a degenerative retinal disease that causes blindness in people 60-65 years and older, with the highest prevalence appearing in people 90 years-old or more. Epidemiological estimates indicate that the number of cases is increasing, and will almost double in the next 20 years. Preventive measures require precise etiological knowledge. This is quite difficult, since AMD is a multifactorial condition with intricate relationships between causes and risk factors. In this review, we describe the impact of light on the structure and physiology of the retina and the pigment epithelium, taking into account the continuous exposure to natural and artificial light sources along the life of an individual. A large body of experimental evidence demonstrates the toxic effects of some lighting conditions on the retina and the pigment epithelium, and consensus exists about the importance of photo-oxidation phenomena in the causality chain between light and retinal damage. Here, we analyzed the transmission of light to the retina, and compared the aging human macula in healthy and diseased retinas, as shown by histology and non-invasive imaging systems. Finally, we have compared the putative retinal photosensitive molecular structures that might be involved in the genesis of AMD. The relationship between these compounds and retinal damage supports the hypothesis of light as an important initiating cause of AMD.
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Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.
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Purpose: To quantify fundus autofluorescence (qAF) in patients with recessive Stargardt disease (STGD1). Methods: A total of 42 STGD1 patients (ages: 7-52 years) with at least one confirmed disease-associated ABCA4 mutation were studied. Fundus AF images (488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, zero GL, magnification, and normative optical media density to yield qAF. Texture factor (TF) was calculated to characterize inhomogeneities in the AF image and patients were assigned to the phenotypes of Fishman I through III. Results: Quantified fundus autofluorescence in 36 of 42 patients and TF in 27 of 42 patients were above normal limits for age. Young patients exhibited the relatively highest qAF, with levels up to 8-fold higher than healthy eyes. Quantified fundus autofluorescence and TF were higher in Fishman II and III than Fishman I, who had higher qAF and TF than healthy eyes. Patients carrying the G1916E mutation had lower qAF and TF than most other patients, even in the presence of a second allele associated with severe disease. Conclusions: Quantified fundus autofluorescence is an indirect approach to measuring RPE lipofuscin in vivo. We report that ABCA4 mutations cause significantly elevated qAF, consistent with previous reports indicating that increased RPE lipofuscin is a hallmark of STGD1. Even when qualitative differences in fundus AF images are not evident, qAF can elucidate phenotypic variation. Quantified fundus autofluorescence will serve to establish genotype-phenotype correlations and as an outcome measure in clinical trials.
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Purpose: Bisretinoids form in photoreceptor cells and accumulate in retinal pigment epithelium (RPE) as lipofuscin. To examine the role of these fluorophores as mediators of retinal light damage, we studied the propensity for light damage in mutant mice having elevated lipofuscin due to deficiency in the ATP-binding cassette (ABC) transporter Abca4 (Abca4(-/-) mice) and in mice devoid of lipofuscin owing to absence of Rpe65 (Rpe65(rd12)). Methods: Abca4(-/-), Rpe65(rd12), and wild-type mice were exposed to 430-nm light to produce a localized lesion in the superior hemisphere of retina. Bisretinoids of RPE lipofuscin were measured by HPLC. In histologic sections, outer nuclear layer (ONL) thickness was measured as an indicator of photoreceptor cell degeneration, and RPE nuclei were counted. Results: As shown previously, A2E levels were increased in Abca4(-/-) mice. These mice also sustained light damage-associated ONL thinning that was more pronounced than in age-matched wild-type mice; the ONL thinning was also greater in 5-month versus 2-month-old mice. Numbers of RPE nuclei were reduced in light-stressed mice, with the reduction being greater in the Abca4(-/-) than wild-type mice. In Rpe65(rd12) mice bisretinoid compounds of RPE lipofuscin were not detected chromatographically and light damage-associated ONL thinning was not observed. Conclusions: Abca4(-/-) mice that accumulate RPE lipofuscin at increased levels were more susceptible to retinal light damage than wild-type mice. This finding, together with results showing that Rpe65(rd12) mice did not accumulate lipofuscin and did not sustain retinal light damage, indicates that the bisretinoids of retinal lipofuscin are contributors to retinal light damage.
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We propose a method of analyzing spatiotemporal data by decomposition into deterministic nonparametric functions of time and space, linear functions of other covariates, and a random component that is spatially, though not temporally, correlated. The resulting model is used for spatial interpolation and especially for estimation of a spatially dependent temporal average. The results are applied to part of the PM2.5 network established by the U.S. Environmental Protection Agency, covering three southeastern U.S. states. A novel feature of the analysis is a variant of the expectation-maximization algorithm to account for missing data. The results show, among other things, that a substantial part of the region is in violation of the proposed long-term average standard for PM2.5.
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Purpose: To investigate fundus autofluorescence (AF) characteristics in the Abca4(-/-) mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments. Methods: Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4(-/-) mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups. Results: In Abca4(-/-) mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4(-/-) mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4(-/-) mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4(-/-) mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months. Conclusions: Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function.
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To evaluate the feasibility and reliability of a standardized approach for quantitative measurements of fundus autofluorescence (AF) in images obtained with a confocal scanning laser ophthalmoscope (cSLO). AF images (30°) were acquired in 34 normal subjects (age range, 20-55 years) with two different cSLOs (488-nm excitation) equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, the zero GL, and the magnification, to give quantified autofluorescence (qAF). Images from subjects and fixed patterns were used to test detector linearity with respect to fluorescence intensity, the stability of qAF with change in detector gain, field uniformity, effect of refractive error, and repeatability. qAF was independent of detector gain and laser power over clinically relevant ranges, provided that detector gain was adjusted to maintain exposures within the linear detection range (GL < 175). Field uniformity was better than 5% in a central 20°-diameter circle but decreased more peripherally. The theoretical inverse square magnification correction was experimentally verified. Photoreceptor bleaching for at least 20 seconds was performed. Repeatability (95% confidence interval) for same day and different-day retests of qAF was ±6% to ±14%. Agreement (95% confidence interval) between the two instruments was <11%. Quantitative AF imaging appears feasible. It may enhance understanding of retinal degeneration, serve as a diagnostic aid and as a sensitive marker of disease progression, and provide a tool to monitor the effects of therapeutic interventions.
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By its action on rhodopsin, light triggers the well-known visual transduction cascade, but can also induce cell damage and death through phototoxic mechanisms - a comprehensive understanding of which is still elusive despite more than 40 years of research. Herein, we integrate recent experimental findings to address several hypotheses of retinal light damage, premised in part on the close anatomical and metabolic relationships between the photoreceptors and the retinal pigment epithelium. We begin by reviewing the salient features of light damage, recently joined by evidence for retinal remodeling which has implications for the prognosis of recovery of function in retinal degenerations. We then consider select factors that influence the progression of the damage process and the extent of visual cell loss. Traditional, genetically modified, and emerging animal models are discussed, with particular emphasis on cone visual cells. Exogenous and endogenous retinal protective factors are explored, with implications for light damage mechanisms and some suggested avenues for future research. Synergies are known to exist between our long term light environment and photoreceptor cell death in retinal disease. Understanding the molecular mechanisms of light damage in a variety of animal models can provide valuable insights into the effects of light in clinical disorders and may form the basis of future therapies to prevent or delay visual cell loss.
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To evaluate Stargardt disease (STGD) progression and relative lipofuscin levels via autofluorescence image analysis. The relationship between focally increased autofluorescence (FIAF), geographic atrophy (GA) and focally decreased autofluorescence (FDAF) was analyzed in serial, registered autofluorescence (AF) scans of 10 patients with STGD (20 eyes, 40 scans; mean follow-up, 2.0 years) using automated techniques. GA progressed uniformly in a transition zone with minimal FIAF. Only 4.3% of FIAF progressed to GA or FDAF, despite significant progression of GA (median 30%/year) and FDAF (mean, 29%/year). As a spatial predictor, the mean chance of FIAF for progression to FDAF was 4.3% +/- 4.4%, significantly less than that of random areas (6.7% +/- 4.0%, P = 0.029, Mann-Whitney test). In the seven eyes with GA, the mean chance of FIAF in the transition zone for transition to GA was 12% +/- 8.9%, significantly less than that of random areas (33% +/- 3.6%, P = 0.026, Mann-Whitney test). Autofluorescent flecks and FIAF deposits with AF levels elevated above the initial macular background were less likely in the short term (2 years) to transform to GA and FDAF (AF levels below the final background) than random areas, suggesting additional mechanisms beyond direct lipofuscin toxicity. FIAF/FDAF levels were observed to fluctuate, with focal remodeling of FIAF and FDAF, or rarely, even transition of FDAF to FIAF. FDAF tended to develop, not coincident with, but adjacent to initial FIAF. Because AF identifies these characteristic biological markers so specifically, autofluorescence metrics merit consideration in the study of STGD.
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A GROWING body of literature attests to the deleterious effects of long term exposure to light1-8. To define more critically the differences between thermal and photochemical effects, we have exposed the retinae of rhesus monkeys to eight monochromatic laser lines from 1,064-441.6 nm. Thermal damage to the retina is to be expected for the 1,064-nm line since the photopigments are not involved and energy absorption takes place predominantly in the melanin granules of the pigment epithelium and the choroid. Although data on pathogenesis are not yet available, we found some interesting differences in retinal sensitivity in going from the near infrared to the blue wavelengths in the visible spectrum.
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Stargardt disease (STGD, also known as fundus flavimaculatus; FFM) is an autosomal recessive retinal disorder characterized by a juvenile-onset macular dystrophy, alterations of the peripheral retina, and subretinal deposition of lipofuscin-like material. A gene encoding an ATP-binding cassette (ABC) transporter was mapped to the 2-cM (centiMorgan) interval at 1p13-p21 previously shown by linkage analysis to harbour the STGD gene. This gene, ABCR, is expressed exclusively and at high levels in the retina, in rod but not cone photoreceptors, as detected by in situ hybridization. Mutational analysis of ABCR in STGD families revealed a total of 19 different mutations including homozygous mutations in two families with consanguineous parentage. These data indicate that ABCR is the causal gene of STGD/FFM.
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Outer segments of mammalian rod photoreceptor cells contain an abundantly expressed membrane protein that migrates with an apparent molecular mass of 220 kDa by SDS-gel electrophoresis. We have purified the bovine protein by immunoaffinity chromatography, determined its primary structure by cDNA cloning and direct peptide sequence analysis, and mapped its distribution in photoreceptors by immunocytochemical and biochemical methods. The full-length cDNA encodes a 2280-amino acid protein (calculated molecular mass of 257 kDa) consisting of two structurally related, tandem arranged halves. Each half consists of a hydrophobic domain containing six putative transmembrane segments followed by an ATP-binding cassette. A data base homology search showed that the rod outer segment 220-kDa protein is 40-50% identical in amino acid sequence to the ABC1 and ABC2 proteins cloned from a mouse macrophage cell line. Photoaffinity labeling with 8-azido-ATP and nucleotide inhibition studies confirmed that both ATP and GTP bind to this protein with similar affinities. Concanavalin A labeling and endoglycosidase H digestion indicated that the rod outer segment protein contains at least one carbohydrate chain. Immunocytochemical and biochemical studies have revealed that the 220-kDa glycoprotein is distributed along the rim region and incisures of rod outer segment disc membranes. From these studies we conclude that the 220-kDa glycoprotein of bovine rod outer segment disc membranes or Rim ABC protein is a new member of the superfamily of ABC transporters and is the mammalian homolog of the frog photoreceptor rim protein.
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Increased accumulation of lipofuscin in cells of the retinal pigment epithelium (RPE) is seen in several forms of macular degeneration, a common cause of blindness in humans. A major fluorophore of lipofuscin is the toxic bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). Previously, we generated mice with a knockout mutation in the abcr gene. This gene encodes rim protein (RmP), an ATP-binding cassette transporter in rod outer segments. Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over controls. Here, we identify three precursors of A2E in ocular tissues from abcr-/- mice and humans with ABCR-mediated recessive macular degenerations. Our results corroborate the scheme proposed by C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon & J. Sparrow [Proc. Natl. Acad. Sci. USA (1998) 95, 14609-14613], for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-trans-RAL) with phosphatidylethanolamine to form a Schiff base; (ii) condensation of the amine product with a second all-trans-RAL to form a bis-retinoid; (iii) oxidation to yield a pyridinium salt; and (iv) hydrolysis of the phosphate ester to yield A2E. The latter two reactions probably occur within RPE phagolysosomes. As predicted by this model, formation of A2E was completely inhibited when abcr-/- mice were raised in total darkness. Also, once formed, A2E was not eliminated by the RPE. These data suggest that humans with retinal or macular degeneration caused by loss of RmP function may slow progression of their disease by limiting exposure to light. The precursors of A2E identified in this study may represent pharmacological targets for the treatment of ABCR-mediated macular degeneration.
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Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.
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To determine whether lipofuscin is detrimental to lysosomal and antioxidant function in cultured human retinal pigment epithelial (RPE) cells. Isolated lipofuscin granules were fed to confluent RPE cultures and the cells maintained in basal medium for 7 days. Parallel cultures were established that did not receive lipofuscin. Cultures were either exposed to visible light (390-550 nm) at an irradiance of 2.8 mW/cm(2) or maintained in the dark at 37 degrees C for up to 24 hours. Cells were subsequently assessed for cell viability, lysosomal enzyme activity, and antioxidant capacity. There was no loss of cell viability during the first 3 hours of light exposure, whereas a 10% loss of viability was observed in lipofuscin-fed cultures after 6 hours' exposure to light. Activities of acid phosphatase, N-acetyl-beta-glucuronidase, and cathepsin D were decreased by up to 50% in lipofuscin-fed cells exposed to light compared with either unfed cells or cells maintained in the dark. There was also a decrease in the antioxidant potential of RPE cells. Catalase and superoxide dismutase activities decreased by up to 60% and glutathione levels by 28% in light-exposed lipofuscin-fed cells compared with unfed cells or cells maintained in the dark. Lipofuscin has the capacity to reduce the efficacy of the lysosomal and antioxidant systems in RPE cells that may play an important role in retinal ageing and the development of age-related macular degeneration.
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The hallmarks of age-related macular degeneration, the leading cause of blindness in the developed world, are the subretinal deposits known as drusen. Drusen identification and measurement play a key role in clinical studies of this disease. Current manual methods of drusen measurement are laborious and subjective. Our purpose was to expedite clinical research with an accurate, reliable digital method. An interactive semi-automated procedure was developed to level the macular background reflectance for the purpose of morphometric analysis of drusen. 12 color fundus photographs of patients with age-related macular degeneration and drusen were analyzed. After digitizing the photographs, the underlying background pattern in the green channel was leveled by an algorithm based on the elliptically concentric geometry of the reflectance in the normal macula: the gray scale values of all structures within defined elliptical boundaries were raised sequentially until a uniform background was obtained. Segmentation of drusen and area measurements in the central and middle subfields (1000 microm and 3000 microm diameters) were performed by uniform thresholds. Two observers using this interactive semi-automated software measured each image digitally. The mean digital measurements were compared to independent stereo fundus gradings by two expert graders (stereo Grader 1 estimated the drusen percentage in each of the 24 regions as falling into one of four standard broad ranges; stereo Grader 2 estimated drusen percentages in 1% to 5% intervals). The mean digital area measurements had a median standard deviation of 1.9%. The mean digital area measurements agreed with stereo Grader 1 in 22/24 cases. The 95% limits of agreement between the mean digital area measurements and the more precise stereo gradings of Grader 2 were -6.4 % to +6.8 % in the central subfield and -6.0 % to +4.5 % in the middle subfield. The mean absolute differences between the digital and stereo gradings 2 were 2.8 +/- 3.4% in the central subfield and 2.2 +/- 2.7% in the middle subfield. Semi-automated, supervised drusen measurements may be done reproducibly and accurately with adaptations of commercial software. This technique for macular image analysis has potential for use in clinical research.
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Mutations in ABCA4, which encodes a photoreceptor specific ATP-binding cassette transporter (ABCR), cause autosomal recessive forms of human blindness due to retinal degeneration (RD) including Stargardt disease. The exact disease sequence leading to photoreceptor and vision loss in ABCA4-RD is not known. Extrapolation from murine and in vitro studies predicts that two of the earliest pathophysiological features resulting from disturbed ABCR function in man would be slowed kinetics of the retinoid cycle and accelerated deposition of lipofuscin in the retinal pigment epithelium (RPE). To determine the human pathogenetic sequence, we studied surrogate measures of retinoid cycle kinetics, lipofuscin accumulation, and rod and cone photoreceptor and RPE loss in ABCA4-RD patients with a wide spectrum of disease severities. There were different extents of photoreceptor/RPE loss and lipofuscin accumulation in different regions of the retina. Slowing of retinoid cycle kinetics was not present in all patients; when present, it was not homogeneous across the retina; and the extent of slowing correlated well with the degree of degeneration. The orderly relationship between these phenotypic features permitted the development of a model of disease sequence in ABCA4-RD. The model predicted lipofuscin accumulation as a key and early component of the disease expression in man, as in mice. In man, however, abnormal slowing of the rod and cone retinoid cycle occurs at later stages of the disease sequence. Knowledge of the human ABCA4 disease sequence will be critical for defining rates of progression, selecting appropriate patients and retinal locations for future therapy, and choosing appropriate treatment outcomes.
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Recessive Stargardt's macular degeneration is a blinding disease of children caused by mutations in the ABCA4 (ABCR) gene. Mice with a knockout mutation in abcr accumulate toxic lipofuscin pigments in ocular tissues, similar to affected humans. The major fluorophore of lipofuscin is the bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). In the current study, we sought to define the effect of increasing light on A2E accumulation. We crossed the abcr(-/-) mutation onto an albino background. The retinoid profiles in albino mice indicated higher retinal illuminance than in pigmented mice exposed to similar ambient light. Unexpectedly, A2E levels were not higher in the albino mice. Also, A2E levels in abcr(-/-) mice reared under cyclic light at 30, 120, or 1,700 lux were similar. Thus, increased retinal illuminance was not correlated with higher A2E. A2E has been shown to undergo light-dependent oxidation to yield a series of A2E epoxides or oxiranes. These oxiranes react with DNA in vitro, suggesting a potential mechanism for A2E cytotoxicity. We analyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry. Unlike A2E, the oxiranes were more abundant in albino vs. pigmented abcr(-/-) mice, and in abcr(-/-) mice exposed to increasing ambient light. These observations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by light. Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr(-/-) mice with Accutane (isotretinoin), an inhibitor of rhodopsin regeneration.
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Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390-550 nm at 2.8 milliwatts/cm2) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis.
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To develop mathematical and geometric models of the nonuniform autofluorescence (AF) patterns of foveas of normal subjects and to reconstruct these models from limited subsets of data. Confocal scanning laser ophthalmoscope (cSLO) AF fundus images of normal maculae were obtained from both eyes of 10 middle-aged subjects. They were filtered and contrast enhanced, to obtain elliptical isobars of equal gray levels (GLs) and determine the isobars' resolutions, eccentricities, and angles of orientation. The original image data were fit with a mathematical model of elliptic quadratic polynomials in two equal zones: the center and the remaining annulus. The AF images segmented into nested concentric GL isobars with GLs that increased radially from the least-fluorescent center. The mean isobar resolution was 31 +/- 7 mum. The geometric eccentricity of the ellipses increased from 0.42 +/- 0.12 centrally to 0.52 +/- 0.14 peripherally (P = 0.0005), with mean axes of orientation peripherally 97.12 +/- 15.46 degrees . The model fits to the complete image data had mean absolute normalized errors ranging from 3.6% +/- 3.7% to 7.3% +/- 7.1%. The model fits to small subsets (1% to 2% of total image data) had mean absolute errors ranging from 3.7% +/- 3.8% to 7.3% +/- 7.2%. Normal AF fundus images show finely resolved, concentric, elliptical foveal patterns consistent with the anatomic distribution of fluorescent lipofuscin, light-attenuating macular pigment (MP), cone photopigment, and retinal pigment epithelial (RPE) pigment in the fovea. A two-zone, elliptic, quadratic polynomial model can accurately model foveal data. This model may be useful for image analysis and for automated segmentation of pathology.
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To assess the relationships of drusen, pigment, and focally increased autofluorescence (FIAF) and the reticular pattern of hypoautofluorescence, to distinguish the combined photographic and AF characteristics of early, atrophic, and high-risk fellow eyes in AMD. In a retrospective interinstitutional clinical study, AF and color photograph pairs of 221 eyes were examined: 166 eyes of 83 patients with bilateral large, soft drusen, with and without geographic atrophy (GA), and 55 fellow eyes of 55 patients with unilateral choroidal neovascularization (CNV). Forty-two eyes (one eye from each of 42 patients with early or atrophic AMD) were divided into four groups: 14 with drusen only, 9 with drusen and pigment abnormalities, 11 fellow eyes of patients with unilateral GA, and 8 eyes of patients with bilateral GA (acronyms for the groups: D-D, D-Pig, D-GA and GA-GA, respectively). The 55 fellow eyes of patients with CNV were divided into three groups: 19 eyes with no FIAF (CNV-0), 16 with FIAF without reticular AF (CNV-1), and 20 eyes with reticular AF and/or pseudodrusen (CNV-R). Image pairs of eyes with FIAF were registered, and drusen, pigment, and FIAF were segmented using automated background leveling and thresholding. All 221 eyes were surveyed for reticular AF and reticular pseudodrusen. The main outcome measures were (1) the fraction and relative probability of FIAF colocalizing with drusen and pigment and (2) the presence or absence of reticular AF and reticular pseudodrusen. The mean fractions of FIAF that colocalized with large drusen were: D-D group, 0.46 +/- 0.21; D-Pig group, 0.42 +/- 0.29; D-GA group, 0.13 +/- 0.09; and GA-GA group, 0.11 +/- 0.12. Comparisons between groups showed significant differences when comparing either the D-D group or the D-Pig group with either the D-GA group or the GA-GA group (P between 0.0001 and 0.015), whereas other comparisons were nonsignificant (Mann-Whitney rank sum test). The mean probabilities of FIAF colocalizing with large drusen relative to chance (1.0) were: D-D group, 4.7 +/- 2.5; D-Pig group, 4.3 +/- 2.3; D-GA group, 1.4 +/- 0.8; and GA-GA group, 1.8 +/- 1.3, with similar significant differences as for the colocalization fractions. The mean probability of FIAF colocalizing with small to intermediate drusen in the D-D group was 1.5 +/- 1.3, which was not significantly different from chance. In the D-Pig group, the median probability of FIAF colocalizing with pigment abnormalities was 10.0 (range, 1.1-51.0). The AF patterns in 15 of 19 eyes in the CNV-0 group were normal; the remainder had nonreticular hypoautofluorescence only. In the CNV-1 group, the relations of FIAF with drusen and pigment were similar to those in the early AMD groups. CNV-R comprised 20 of 55 eyes in the CNV group, but reticular autofluorescence and/or pseudodrusen were found in only 14 of 166 eyes of the early and atrophic groups. Of the 34 total eyes with reticular AF or pseudodrusen, 28 had both, 4 had reticular AF only, and 2 had reticular pseudodrusen only. There are clear relationships between AF patterns and clinical AMD status. In early AMD, FIAF's colocalization with large, soft drusen and hyperpigmentation is several times greater than chance, suggesting linked disease processes. In advanced atrophic AMD, FIAF is found mostly adjacent to drusen and GA, suggesting that dispersal of drusen-associated lipofuscin is a marker of atrophic disease progression. In the neovascular case, a large group of fellow eyes have no FIAF abnormalities, suggesting that lipofuscin is not a major determinant of CNV. However, reticular hypoautofluorescence, consistent with widespread inflammatory damage to the RPE, appears to be a highly sensitive imaging marker for the disease that determines reticular pseudodrusen and is strongly associated with CNV.
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Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging. Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square (1/2) degrees per side. Pre- and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2 degrees field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated. Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed. The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.
Article
To describe the phenotype and genotype of patients with early-onset Stargardt disease. Retrospective cohort study. Fifty-one Stargardt patients with age at onset ≤10 years. We reviewed patient medical records for age at onset, medical history, initial symptoms, best-corrected visual acuity (BCVA), ophthalmoscopy, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography (ffERG). The ABCA4 gene was screened for mutations. Age at onset, BCVA, fundus appearance, FAF, FA, SD-OCT, ffERG, and presence of ABCA4 mutations. The mean age at onset was 7.2 years (range, 1-10). The median times to develop BCVA of 20/32, 20/80, 20/200, and 20/500 were 3, 5, 12, and 23 years, respectively. Initial ophthalmoscopy in 41 patients revealed either no abnormalities or foveal retinal pigment epithelium (RPE) changes in 10 and 9 patients, respectively; the other 22 patients had foveal atrophy, atrophic RPE lesions, and/or irregular yellow-white fundus flecks. On FA, there was a "dark choroid" in 21 out of 29 patients. In 14 out of 50 patients, foveal atrophy occurred before flecks developed. On FAF, there was centrifugal expansion of disseminated atrophic spots, which progressed to the eventual profound chorioretinal atrophy. Spectral-domain OCT revealed early photoreceptor damage followed by atrophy of the outer retina, RPE, and choroid. On ffERG in 26 patients, 15 had normal amplitudes, and 11 had reduced photopic and/or scotopic amplitudes at their first visit. We found no correlation between ffERG abnormalities and the rate of vision loss. Thirteen out of 25 patients had progressive ffERG abnormalities. Finally, genetic screening of 44 patients revealed ≥2 ABCA4 mutations in 37 patients and single heterozygous mutations in 7. In early-onset Stargardt, initial ophthalmoscopy can reveal no abnormalities or minor retinal abnormalities. Yellow-white flecks can be preceded by foveal atrophy and may be visible only on FAF. Although ffERG is insufficient for predicting the rate of vision loss, abnormalities can develop. Over time, visual acuity declines rapidly in parallel with progressive retinal degeneration, resulting in profound chorioretinal atrophy. Thus, early-onset Stargardt lies at the severe end of the spectrum of ABCA4-associated retinal phenotypes. Copyright © 2014 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.
Article
The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid by-product of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of this study was to correlate the distribution of lipofuscin and A2E across the human RPE. Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on both flat-mounted RPE tissue sections and on retinal cross sections. Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased towards the central region. High-resolution MALDI-IMS of retinal cross sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. This report is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.
Article
High doses of light can cause damage to the retina, e.g. during intraocular surgery. Previously, thiols have been demonstrated to protect against retinal damage in various damage models. Such protection is very promising for clinical practice. Retinal light damage can be caused by a relatively short exposure to high irradiance levels. These conditions occur during intraocular surgery. In the current study we therefore investigated whether the thiol N-acetylcysteine protects against retinal light damage under high irradiance conditions in the rat retina. Two stereoisomers of this thiol were tested for protection against two spectrally denned types of retinal light damage. Shortly after administration N-acetyl-L-cysteine in doses of 270–1000 mg/kg intraperitoneal protected against 380 nm (UVA) light but not against 470 nm (blue) light. Two hours after injection the protection had diminished. We observed no protection by the stereoisomer N-acetyl-D-cysteine. From this study we conclude that N-acetyl-L-cysteine protects stereospecifically against retinal damage in the UV but not in the visible part of the spectrum. This limits the possible applications.
Article
In replyWe agree with Smith that the subject of hyperautofluorescent changes in Stargardt disease is an interesting one that may be illuminating to understanding underlying disease pathophysiology. In our report, we had examined longitudinal changes in fundus autofluorescence over a series of time points in a number of affected eyes. Our observations describe a consistent pattern of temporal and spatial progression in the flecks associated with Stargardt disease. The general pattern was one in which brighter hyperautofluorescent flecks move centrifugally from the center of the macula. As these flecks move outward, they leave in their wake darker, hypoautofluorescent lesions. As a result, fundus autofluorescence at a particular point in the fleck's path may be described to be first increasing with time, becoming increasingly hyperautofluorescent relative to background, then decreasing back to background levels, and subsequently decreasing further to become hypoautofluorescent relative to background. We agree with Smith that within the time of our study (11-57 months), we did not observe further decreases in fundus autofluorescence in the tracks left behind by progressing flecks that may be described as frank atrophy (ie, the confluent absence of autofluorescence signal suggestive of retinal pigment epithelial cell loss).
Article
The recent article by Cukras et al1 that demonstrated interesting centrifugal “trajectories” for hyperautofluorescent flecks in Stargardt disease enhances our understanding of the progression of this disease and bears on fundamental questions about the role of lipofuscin. Given the importance of these issues, I suggest that the reference therein to related quantitative work2 should also be amplified. We specifically investigated the spatial predictive value of hyperautofluorescent lesions for the subsequent development of hypoautofluorescence or atrophy in the same location with precise registration and segmentation of serial images as well as statistical calculation of spatial predictive values. To our knowledge, this is the only such study, and the results were surprising. If toxic effects to the retinal pigment epithelium by excess lipofuscin drives Stargardt disease, then flecks, with high lipofuscin levels, should be more likely to turn atrophic than neighboring background. However, the predictive value of hyperautofluorescent lesions for future hypoautofluorescence or atrophy at a given location was actually less than background, suggesting that an elevated level of lipofuscin, while an important marker for Stargardt disease, may not be a major cause.
Chapter
When the adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, ABCA4 (originally named ABCR), was cloned and characterized in 1997 as the causal gene for autosomal recessive Stargardt disease (arSTGD or STGD1) (1) it seemed as if just another missing link was added to the extensive table of genetic determinants of rare monogenic retinal dystrophies. Now, 9 yr later, the ABCA4 gene continues to emerge as the predominant determinant of a wide variety of retinal degeneration phenotypes. ABCA4 has caused exciting and sometimes intense discussions among ophthalmologists and geneticists, resulting in more than 150 publications during this time.
Article
To evaluate retinal pigment epithelial (RPE) atrophy in patients with Stargardt disease using autofluorescence imaging (AF). Retrospective observational case series. Demographics, best-corrected visual acuity (BCVA), AF images, and electrophysiology responses (group 1, macular dysfunction; group 2, macula + cone dysfunction; group 3, macula + cone-rod dysfunction) were evaluated at presentation and follow-up in a group of 12 patients (24 eyes) with Stargardt disease. The existence, development, and rate of enlargement of areas of RPE atrophy over time were evaluated using AF imaging. A linear regression model was used to investigate the effects of AF and electrophysiology on rate of atrophy enlargement and BCVA, adjusting for age of onset and duration of disease. Eight male and 4 female patients (median age 42 years; range 24-69 years) were followed for a median of 41.5 months (range 13-66 months). All 12 patients had reduced AF compatible with RPE atrophy at presentation and in all patients the atrophy enlarged during follow-up. The mean rate of atrophy enlargement for all patients was 1.58 mm(2)/y (SD 1.25 mm(2)/y; range 0.13-5.27 mm(2)/y). Only the pattern of functional loss present as detected by electrophysiology was statistically significantly associated with the rate of atrophy enlargement when correcting for other variables (P < .001), with patients in group 3 (macula + cone-rod dysfunction) having the fastest rate of atrophy enlargement (1.97 mm(2)/y, SD 0.70 mm(2)/y) (group 1 [macula] 1.09 mm(2)/y, SD 0.53 mm(2)/y; group 2 [macula + cone] 1.89 mm(2)/y, SD 2.27 mm(2)/y). Variable rates of atrophy enlargement were observed in patients with Stargardt disease. The pattern of functional loss detected on electrophysiology was strongly associated with the rate of atrophy enlargement over time, thus serving as the best prognostic indicator for patients with this inherited retinal disease.
Article
The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber's congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy.
Article
We tested for protection of blue light-exposed A2E-containing retinal pigment epithelial (RPE) from damage through the implementation of polycarbonate filters containing varying levels of a pigment that absorbs short wavelength light. Human adult RPE cells (ARPE-19) that had accumulated synthesized A2E were exposed to either a light line delivered from a tungsten halogen source (430 ± 20 nm; 8 mW/cm) or to the entire area of a 35 mm dish (1 mW/cm). Blue-light absorbing polycarbonate filters (2.5 × 4 cm) containing varying levels of short-wavelength light absorbing pigment (1.0, 1.9, 3.8, 7.5, 15, and 35 ppm) or no dye (PC) were placed in the light path. Cytotoxicity was measured by the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric microtiter assay (Roche Diagnostics Corporation, Indianapolis, IN) or by fluorescent staining of non-viable cells. When filters containing blue-light absorbing dye were placed in the light path, protection of 430 nm irradiated A2E-laden RPE was observed. The extent of protection was dependent on the concentration of the dye. By MTT assay and fluorescence labeling, statistically significant differences (p < 0.05) between irradiation in the absence of a filter and irradiation in the presence of a filter were observed. The series of filters tested in this work provided protection against blue light damage in a culture model.
Article
The photopathology of retinal lesions produced by extended exposure (1000 sec) to low corneal power levels (62 microW) of blue light (441 nm) was investigated by light microscopy in 20 rhesus eyes over an interval ranging from 1 hr to 90 days after exposure. Results indicate a nonthermal type of photochemical lesion originating in the retinal pigment epithelium and leading to a histological response with hypopigmentation which requires 48 hr to appear. This type of lesion helps to explain solar retinitis and eclipse blindness and has significance for aging and degenerative changes in the retina.
Article
Extended exposure (100s) of the macaque retina to blue light (400-500nm) produces a photochemical type or types of lesion. The basic mechanisms responsible for such photic damage are unknown but the toxic combination of light and oxygen leading to the free radicals O-.2, H2O2, OH., and O2(1 delta) have been suggested as a possible source of the phototoxicity. To test this hypothesis, the radiant exposure (J. cm-2) to short wavelength light (435-445nm) required for minimal damage in the macaque retina is under investigation as a function of oxygenation and after administration of substances known to either inhibit/scavenge radicals or act as anti-inflammatory/anti-oxidant agents. Substances under study include beta-carotene, steroids, catalase and SOD. Here we report radiant exposure in J.cm-2 needed to produce a minimal lesion vs oxygenation as measured by partial pressure of O2 in arterial blood (Po2). There is a sharp drop in the radiant exposure threshold with increase in the partial pressure of O2 in arterial blood, e.g. 30 J.cm-2 at 75 torr to 10 J.cm-2 at 271 torr, a factor of 3. Methylprednisolone injected intravenously one hour before exposure (125 mg) has been shown to raise the threshold for retinal damage in two macaques by a factor of approximately 2. Another animal fed beta-carotene (7.5 mg daily) over a period of 3 months has been exposed to blue light at several levels of oxygenation. The results suggest a protective effect.
Article
The historical development of the idea that light deprivation could have therapeutic benefit for patients with retinitis pigmentosa is reviewed. A patient with autosomal recessive and a patient with autosomal dominant retinitis pigmentosa participated in a trial of light deprivation of one eye with an opaque scleral contact lens for 5 yr for about 6–8 hr per day. Each showed comparable progression in both eyes as monitored by changes in visual acuity, visual fields, fundus appearance, and electroretinograms (ERGs).In addition to the well known abnormality in dark adaptation with consequent night blindness, studies of cone ERG b-wave implicit times have shown that patients with early retinitis pigmentosa can have a defect in the mechanism by which their cone system adapts to light. Until more is known about this defect and the pathogenesis of retinitis pigmentosa, we recommend that patients wear dark sunglasses for outdoor use although we have no evidence at this time to substantiate that light deprivation with any type of sunglass or use of an opaque lens will modify the rates of progression of these diseases.
Article
Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.
Article
Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime ca 7 microseconds) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.
Article
A universal biomarker of cellular ageing in eukaryotic postmitotic cells is the appearance over time of autofluorescent lysosomal residual bodies called age pigments or lipofuscin granules. Their role in the process of cellular ageing has been debated without resolution. Neither the identity nor mechanism of formation of the fluorophores has been definitively determined. A postmitotic cell type that accumulates large quantities of age pigments is the ocular retinal pigment epithelium. We have now identified the major orange-emitting fluorophore of these pigments using fast-atom bombardment tandem mass spectrometry with collisional activation analysis. It is an amphoteric quaternary amine that arises as a Schiff base reaction product of retinaldehyde and ethanolamine. This compound should display lysosomotropic detergent behaviour which would help explain many of the age-related changes shown in this cell. These results suggest a new role for Schiff base reaction products as lysosomotropic amines in the genesis of cellular age pigments.
Article
Rim protein (RmP) is a high-Mr membrane glycoprotein that has been localized to the rims of photoreceptor outer segment discs, but its molecular identity is unknown. Here, we describe the purification of RmP and present the sequence of its mRNA. RmP is a new member of the ATP-binding cassette (ABC) transporter superfamily. We show that RmP is expressed specifically in photoreceptors and predominantly in outer segments. Further, RmP is identical to the protein recently shown to be affected in recessive Stargardt's disease. RmP is the first ABC transporter observed in photoreceptors and may play a role in the photoresponse.
Article
Accumulation of lipofuscin (LF) is a prominent feature of aging in the human retinal pigment epithelium (RPE) cells. This age pigment exhibits substantial photoreactivity, which may increase the risk of retinal photodamage and contribute to age-related maculopathy. In a previous study, we detected singlet oxygen generation by lipofuscin granules excited with blue light. In this paper we investigated the ability of hydrophobic components of lipofuscin to photogenerate singlet oxygen in non-polar environments. Singlet oxygen was detected directly by monitoring its characteristic phosphorescence at ca 1270 nm. The action spectrum of singlet oxygen formation indicated that this process was strongly wavelength-dependent and its efficiency decreased with increasing wavelength by a factor of ten, comparing 420 nm and 520 nm. The quantum yield of singlet oxygen increased with increasing concentration of oxygen. Using laser flash photolysis we studied the possible mechanism of singlet oxygen formation. The observed transient, with a broad absorption spectrum peaking at around 440 nm, was identified as a triplet with lifetime ca 11 microseconds. It was quenched by both molecular oxygen and beta-carotene with concomitant formation of a beta-carotene triplet state. These results indicate the potential role of hydrophobic components of lipofuscin in blue light-induced damage to the RPE.
Article
To report the spectrum of ophthalmic findings in patients with Stargardt dystrophy or fundus flavimaculatus who have a specific sequence variation in the ABCR gene. Twenty-nine patients with Stargardt dystrophy or fundus flavimaculatus from different pedigrees were identified with possible disease-causing sequence variations in the ABCR gene from a group of 66 patients who were screened for sequence variations in this gene. Patients underwent a routine ocular examination, including slitlamp biomicroscopy and a dilated fundus examination. Fluorescein angiography was performed on 22 patients, and electroretinographic measurements were obtained on 24 of 29 patients. Kinetic visual fields were measured with a Goldmann perimeter in 26 patients. Single-strand conformation polymorphism analysis and DNA sequencing were used to identify variations in coding sequences of the ABCR gene. Three clinical phenotypes were observed among these 29 patients. In phenotype I, 9 of 12 patients had a sequence change in exon 42 of the ABCR gene in which the amino acid glutamic acid was substituted for glycine (Gly1961Glu). In only 4 of these 9 patients was a second possible disease-causing mutation found on the other ABCR allele. In addition to an atrophic-appearing macular lesion, phenotype I was characterized by localized perifoveal yellowish white flecks, the absence of a dark choroid, and normal electroretinographic amplitudes. Phenotype II consisted of 10 patients who showed a dark choroid and more diffuse yellowish white flecks in the fundus. None exhibited the Gly1961Glu change. Phenotype III consisted of 7 patients who showed extensive atrophic-appearing changes of the retinal pigment epithelium. Electroretinographic cone and rod amplitudes were reduced. One patient showed the Gly1961Glu change. A wide variation in clinical phenotype can occur in patients with sequence changes in the ABCR gene. In individual patients, a certain phenotype seems to be associated with the presence of a Gly1961Glu change in exon 42 of the ABCR gene. The identification of correlations between specific mutations in the ABCR gene and clinical phenotypes will better facilitate the counseling of patients on their visual prognosis. This information will also likely be important for future therapeutic trials in patients with Stargardt dystrophy.
Article
Rim protein (RmP) is an ABC transporter of unknown function in rod outer segment discs. The human gene for RmP (ABCR) is affected in several recessive retinal degenerations. Here, we characterize the ocular phenotype in abcr knockout mice. Mice lacking RmP show delayed dark adaptation, increased all-trans-retinaldehyde (all-trans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE). These data suggest that RmP functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation in discs of the noncovalent complex between opsin and all-trans-RAL. Finally, ABCR-mediated retinal degeneration may result from "poisoning" of the RPE due to A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role.
Article
High doses of light can cause damage to the retina, e.g. during intraocular surgery. Previously, thiols have been demonstrated to protect against retinal damage in various damage models. Such protection is very promising for clinical practice. Retinal light damage can be caused by a relatively short exposure to high irradiance levels. These conditions occur during intraocular surgery. In the current study we therefore investigated whether the thiol N-acetylcysteine protects against retinal light damage under high irradiance conditions in the rat retina. Two stereoisomers of this thiol were tested for protection against two spectrally defined types of retinal light damage. Shortly after administration N-acetyl-L-cysteine in doses of 270-1000 mg/kg intraperitoneally protected against 380 nm (UVA) light but not against 470 nm (blue) light. Two hours after injection the protection had diminished. We observed no protection by the stereoisomer N-acetyl-D-cysteine. From this study we conclude that N-acetyl-L-cysteine protects stereospecifically against retinal damage in the UV but not in the visible part of the spectrum. This limits the possible applications.
Article
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.
Article
A fluorescent component of lipofuscin, A2-E (N-retinylidene-N-retinylethanol-amine) has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. In addition to its lysosomotropic properties A2-E is known to be photoreactive. The purpose of this study was to determine the phototoxic potential of A2-E on RPE cells. A2-E (synthesized by coupling all-trans-retinaldehyde to ethanolamine) was complexed to low-density lipoprotein (LDL) to allow for specific loading of the lysosomal compartment. Human RPE cell cultures were loaded with the A2-E-LDL complex four times within 2 weeks. A2-E accumulation was confirmed by fluorescence microscopy and flow cytometry analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. The phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short wavelength visible light (400-500 nm) and assessing cell viability and lysosomal integrity. Fluorescence microscopy and flow cytometry analysis demonstrated that the intralysosomal accumulation of A2-E in cultured RPE cells increased with the number of feedings. Acridine orange staining confirmed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E-fed cells to light resulted in a significant loss of cell viability by 72 hours, which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity. A2-E is detrimental to RPE cell function by a variety of mechanisms: inhibition of lysosomal degradative capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging as well as retinal diseases associated with excessive lipofuscin accumulation-for example, age-related macular degeneration and Stargardt's disease.
Article
The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage to retinal pigment epithelial (RPE) cells. The purpose of this study was to evaluate caspase-3 and Bcl-2 as executor and modulator, respectively, of the cell death program that is initiated in A2E-containing cells in response to blue light. Human RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light. Caspase-3 activity was assayed by observing cleavage of a fluorogenic peptide substrate, and the effect of a peptide inhibitor of caspase-3 (Z-DEVD-fmk) on the quantity of apoptotic nuclei was determined. ARPE-19 cells were transfected with either a neomycin-selectable expression vector containing Bcl-2 cDNA or a control neomycin-selectable expression vector without Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein expression was confirmed by Western blot analysis. Cell viability was assayed by TdT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidino-2-phenylindole (DAPI) staining and by fluorescence staining of the nuclei of membrane-compromised cells. In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of blue light exposure. The incidence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected with Bcl-2. Blue light illumination of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a proteolytic caspase cascade and that is regulated by Bcl-2.
Article
To examine the ocular phenotype in mice heterozygous for a null mutation in the abcr gene. Retinas and retinal pigment epithelia (RPE) were prepared from wild-type, abcr+/-, and abcr-/- mice. Fresh tissues were homogenized and analyzed by normal phase high-performance liquid chromatography (HPLC) for the presence of retinoids and phospholipids. In another study, fixed tissues were sectioned and analyzed by light and electron microscopy. Finally, anesthetized mice were studied by electroretinography (ERG) at different times after exposure to strong light. A2E, the major fluorophore of lipofuscin, and its precursors, A2PE-H(2) and A2PE, were approximately fourfold more abundant in 8-month-old abcr+/- than in the wild-type retina and RPE. The levels of these substances in abcr+/- mice were approximately 40% those in abcr-/- mice. Lipofuscin pigment-granules were also visible in abcr+/- RPE cells by electron microscopy. Accumulation of A2PE-H(2) and A2E in abcr+/- retina and RPE, respectively, was strongly dependent on light exposure. Heterozygous mutants also exhibited delayed recovery of rod sensitivity by ERG. This delay was correlated with elevated levels of all-trans-retinaldehyde (all-trans-RAL) in retina after a photobleach and was not caused by a reduction in quantum-catch due to depletion of 11-cis-retinaldehyde (11-cis-RAL). Partial loss of the ABCR or rim protein is sufficient to cause a phenotype in mice similar to recessive Stargardt's disease (STGD) and age-related macular degeneration (AMD) in humans. These data are consistent with the suggestion that the STGD carrier-state may predispose to the development of AMD.
Article
The majority of studies on the retina-specific ATP-binding cassette transporter (ABCA4) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4-associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations. Nine well-documented patients representing distinct phenotypes in the continuum of ABCA4-related disorders were selected. All patients received an extensive ophthalmologic evaluation, including kinetic perimetry, fluorescein angiography, and electroretinography (ERG). Mutation analysis had been performed previously with the genotyping microarray (ABCR400 chip) and/or single-strand conformation polymorphism analysis in combination with direct DNA sequencing. In all patients, at least one pathologic ABCA4 mutation was identified. Patient 10034 represented the mild end of the phenotypic spectrum, demonstrating exudative age-related macular degeneration (AMD). Patient 24481 received the diagnosis of late-onset fundus flavimaculatus (FFM), patient 15168 demonstrated the typical FFM phenotype, and patient 19504 had autosomal recessive Stargardt disease (STGD1). Patients 11302 and 7608 exhibited progression from FFM/STGD1 to cone-rod dystrophy (CRD). A more typical CRD phenotype was found in patients 15680 and 12608. Finally, the most severe ABCA4-associated phenotype was retinitis pigmentosa (RP) in patient 11366. This phenotype was characterised by extensive atrophy with almost complete loss of peripheral and central retinal functions. We describe nine patients during different stages of disease progression; together, these patients form a continuum of ABCA4-associated phenotypes. Besides characteristic disorders such as FFM/STGD1, CRD and RP, intermediate phenotypes may be encountered. Moreover, as the disease progresses, marked differences may be observed between initially comparable phenotypes. In contrast, distinctly different phenotypes may converge to a similar final stage, characterised by extensive chorioretinal atrophy and very low visual functions. The identified ABCA4 mutations in most, but not all, patients were compatible with the resulting phenotypes, as predicted by the genotype-phenotype model for ABCA4-associated disorders. With the advent of therapeutic options, recognition by the general ophthalmologist of the various retinal phenotypes associated with ABCA4 mutations is becoming increasingly important.
Article
Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.
following search string in the PubMed database The search was performed on (irradiation[tiab] OR irradiations[tiab] OR photoradiation[tiab] OR photoradiations[tiab] OR radiations[tiab] OR illumination[tiab] OR light[tiab] OR photo[tiab] OR (intraocular[tiab] AND light
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SUPPLEMENTAL MATERIAL AN EXTENSIVE LITERATURE SEARCH WAS PERFORMED USING THE following search string in the PubMed database (available at http://www.ncbi.nlm.nih.gov/). The search was performed on November 2, 2014. (irradiation[tiab] OR irradiations[tiab] OR photoradiation[tiab] OR photoradiations[tiab] OR radiations[tiab] OR illumination[tiab] OR light[tiab] OR photo[tiab] OR (intraocular[tiab] AND light[tiab])) AND ((treat[tiab] OR treatment[tiab] OR treated[tiab]) OR (protect[tiab] OR protection[tiab] OR protected[tiab])