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1.  Light-Induced Retinal Changes Observed with High-Resolution Autofluorescence Imaging of the Retinal Pigment Epithelium 
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 ½° per side. Pre-and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2° 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 method. 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.
PMCID: PMC2790526  PMID: 18408191
2.  In Vivo Autofluorescence Imaging of the Human and Macaque Retinal Pigment Epithelial Cell Mosaic 
Retinal pigment epithelial (RPE) cells are critical for the health of the retina, especially the photoreceptors. A recent study demonstrated that individual RPE cells could be imaged in macaque in vivo by detecting autofluorescence with an adaptive optics scanning laser ophthalmoscope (AOSLO). The current study extended this method to image RPE cells in fixating humans in vivo and to quantify the RPE mosaic characteristics in the central retina of normal humans and macaques.
The retina was imaged simultaneously with two light channels in a fluorescence AOSLO; one channel was used for reflectance imaging of the cones while the other detected RPE autofluorescence. The excitation light was 568 nm, and emission was detected over a 40-nm range centered at 624 nm. Reflectance frames were registered to determine interframe eye motion, the motion was corrected in the simultaneously recorded autofluorescence frames, and the autofluorescence frames were averaged to give the final RPE mosaic image.
In vivo imaging demonstrated that with increasing eccentricity, RPE cell density, and mosaic regularity decreased, whereas RPE cell size and spacing increased. Repeat measurements of the same retinal location 42 days apart showed the same RPE cells and distribution.
The RPE cell mosaic has been resolved for the first time in alert fixating human subjects in vivo using AOSLO. Mosaic analysis provides a quantitative database for studying normal and diseased RPE in vivo. This technique will allow longitudinal studies to track disease progression and assess treatment efficacy in patients and animal models of retinal disease.
PMCID: PMC2790524  PMID: 18952914
3.  Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure 
Many retinal imaging instruments use infrared wavelengths to reduce the risk of light damage. However, we have discovered that exposure to infrared illumination causes a long-lasting reduction in infrared autofluorescence (IRAF). We have characterized the dependence of this effect on radiant exposure and investigated its origin.
A scanning laser ophthalmoscope was used to obtain IRAF images from two macaques before and after exposure to 790-nm light (15-450 J/cm2). Exposures were performed with either raster-scanning or uniform illumination. Infrared autofluorescence images also were obtained in two humans exposed to 790-nm light in a separate study. Humans were assessed with direct ophthalmoscopy, Goldmann visual fields, multifocal ERG, and photopic microperimetry to determine whether these measures revealed any effects in the exposed locations.
A significant decrease in IRAF after exposure to infrared light was seen in both monkeys and humans. In monkeys, the magnitude of this reduction increased with retinal radiant exposure. Partial recovery was seen at 1 month, with full recovery within 21 months. Consistent with a photochemical origin, IRAF decreases caused by either raster-scanning or uniform illumination were not significantly different. We were unable to detect any effect of the light exposure with any measure other than IRAF imaging. We cannot exclude the possibility that changes could be detected with more sensitive tests or longer follow-up.
This long-lasting effect of infrared illumination in both humans and monkeys occurs at exposure levels four to five times below current safety limits. The photochemical basis for this phenomenon remains unknown.
Exposure to infrared illumination at irradiances well below current safety limits can cause a long-lasting decrease in infrared autofluorescence from the retina. It is unclear whether this effect is benign or indicative of a subcellular change that could be cumulatively harmful.
PMCID: PMC4068866  PMID: 24845640
retina; light damage; radiation damage; scanning laser ophthalmoscopy; retinal pigment epithelium
4.  Scanning Laser Ophthalmoscope Measurement of Local Fundus Reflectance and Autofluorescence Changes Arising from Rhodopsin Bleaching and Regeneration 
We measured the bleaching and regeneration kinetics of rhodopsin in the living human eye with two-wavelength, wide-field scanning laser ophthalmoscopy (SLO), and investigated the effect of rhodopsin bleaching on autofluorescence intensity.
The retina was imaged with an Optos P200C SLO by its reflectance of 532 and 633 nm light, and its autofluorescence excited by 532 nm light, before and after exposure to lights calibrated to bleach rhodopsin substantially. Bleaching was confined to circular retinal regions of 4.8° visual angle located approximately 16° superotemporal and superonasal to fixation. Images were captured as 12-bit tiff files and postprocessed to extract changes in reflectance and autofluorescence.
At the locus of bleaching transient increases in reflectance of the 532 nm, but not the 633 nm beam were observed readily and quantified. A transient increase in autofluorescence also occurred. The action spectrum, absolute sensitivity, and recovery of the 532 nm reflectance increase were consistent with previous measurements of human rhodopsin's spectral sensitivity, photosensitivity, and regeneration kinetics. The autofluorescence changes closely tracked the changes in rhodopsin density.
The bleaching and regeneration kinetics of rhodopsin can be measured locally in the human retina with a widely available SLO. The increased autofluorescence excited by 532 nm light upon bleaching appears primarily due to transient elimination of rhodopsin's screening of autofluorescent fluorochromes in the RPE. The spatially localized measurement with a widely available SLO of rhodopsin, the most abundant protein in the retina, could be a valuable adjunct to retinal health assessment.
Rhodopsin was measured locally in the retina with a widely available, dual wavelength scanning laser ophthalmoscope that does not require pupil dilation. Increased autofluorescence attendant bleaching arises largely from transient removal of rhodopsin's screening of autofluorescent fluorochromes.
PMCID: PMC3621503  PMID: 23412087
5.  The susceptibility of the retina to photochemical damage from visible light 
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.
PMCID: PMC3242847  PMID: 22085795
Phototoxicity; Photochemical; Retina; Retinal pigment epithelium; Autofluorescence; Visual cycle; Lipofuscin; Bisretinoids
6.  CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents 
PLoS Medicine  2008;5(2):e39.
In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.
Methods and Findings
We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.
CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.
Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium.
Editors' Summary
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in industrialized countries. The macula is the central region of the retina, the tissue at the back of the eye that detects light and converts it into electrical messages that are sent to the brain. In the commonest form of AMD—“dry” AMD—the light-sensitive cells in the retina (the photoreceptors) gradually die. This degeneration might occur because of damage to the retinal pigment epithelium (RPE). This layer of dark cells lies between the photoreceptors and the choroid, the layer of the eye that contains blood vessels and brings oxygen to the retina. The RPE keeps the retina healthy by transferring the right amount of oxygen and nutrients from the choroid to the retina and by removing worn-out photoreceptor outer segments (the part of the photoreceptor that actually absorbs light) in a process called phagocytosis (engulfment and digestion). In addition to photoreceptor degeneration and RPE shrinkage, a layer of the choroid rich in small blood vessels (the choriocapillaris) also shrinks in dry AMD. For affected individuals, all these changes (which experts describe as retinal atrophy and choroidal involution) mean that the sharp central vision that is needed for reading and driving is destroyed, leaving only dim, burred images or a black hole at the center of the vision.
Why Was This Study Done?
Little is known about the molecular mechanisms that underlie dry AMD and, consequently, there is no cure for it. In this study, the researchers have tested whether a molecule called CD36, which is expressed on the surface of RPE cells, is involved in dry AMD. CD36 is a scavenger receptor—which means it binds many potentially harmful molecules including oxidized fats (which are present in the photoreceptor outer segments) and is involved in their phagocytosis. Phagocytosis itself induces the expression of several proteins in the RPE cells, including COX2, a “proangiogenic” protein that stimulates the growth of blood vessels. Putting this information together, the researchers hypothesized that a defect in CD36 might cause the characteristic retinal atrophy (by preventing the phagocytosis of worn-out photoreceptor outer segments) and choroidal involution (by preventing the induction of COX2 expression and consequently the maintenance of the blood vessels in the choroid) of dry AMD.
What Did the Researchers Do and Find?
The researchers first show that retinal degeneration occurs in rats and mice that express no CD36. This degeneration (which included a reduction in the thickness of the retina, the presence of irregularly shaped photoreceptor outer segments, and the detachment of these structures from the RPE) was seen in old but not young animals. Choroidal involution was also seen in these CD36-deficient animals. This change was present in young mice and rats but increased with age so that by one year old, the choriocapillaris looked moth-eaten. Next, the researchers show that although RPE cells taken from normal animals and grown in dishes were able to make COX2 in response to exposure to purified photoreceptor outer segments, RPE cells from CD36-deficient animals did not. The expression of vascular endothelial growth factor (VEGF; a protein that is needed for normal choroidal development and whose expression is controlled by COX2) showed a similar pattern. Finally, the researchers report that COX2 deficiency in mice caused similar age-dependent choroidal involution and similar effects on VEGF expression in RPE cells as CD36 deficiency.
What Do These Findings Mean?
These findings show that CD36 deficiency leads to progressive, age-related degeneration of photoreceptors and choroidal involution in rats and mice. They also show that CD36 deficiency causes this choroidal involution, the key feature of dry AMD, because it leads to down-regulation of COX2 expression (and subsequently reduced VEGF expression) in the RPE. Researchers now need to find out whether this mechanism for the development of dry AMD holds in people—what happens in animals does not necessarily happen in people. If it does, pharmacological activation of CD36 or restoration of CD36 expression in the RPE might eventually provide a way to treat dry AMD.
Additional Information.
Please access these Web sites via the online version of this summary at
MedlinePlus provides links to information on macular degeneration and an encyclopedia page on macular degeneration (in English and Spanish)
Pages on the US National Institutes of Health NIH SeniorHealth site provides text and spoken information about AMD
The US National Eye Institute and the UK Royal National Institute of Blind People also provide information about AMD
Wikipedia has pages on the retina, photoreceptor cells, retinal pigment epithelium, and choroid (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2245984  PMID: 18288886
7.  Mitochondrial Oxidative Stress in the Retinal Pigment Epithelium Leads to Localized Retinal Degeneration 
Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer.
Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2flox/floxVMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2flox/flox mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology.
Following doxycycline induction of cre, Sod2flox/flox cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2flox/flox cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes.
Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.
The RPE-specific deletion of mitochondrial superoxide dismutase in mice leads to oxidative stress in the RPE, accumulation of autofluorescent material, a decline in the ERG response, and localized death of photoreceptors. This model may be useful in understanding the mechanism of geographic atrophy.
PMCID: PMC4112607  PMID: 24985474
retinal degeneration; mouse model; reactive oxygen species; manganese superoxide dismutase; retinal pigment epithelium
8.  The Bisretinoids of Retinal Pigment Epithelium 
The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL-4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.
PMCID: PMC3288746  PMID: 22209824
A2E; all-trans-retinal; bisretinoid; retinal pigment epithelium; macular degeneration; retina
9.  Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation 
PLoS Medicine  2007;4(6):e230.
RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).
Methods and Findings
RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.
Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.
The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients.
Editors' Summary
The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time.
Why Was This Study Done?
Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision.
What Did the Researchers Do and Find?
The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals.
What Do These Findings Mean?
The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA.
Additional Information.
Please access these Web sites via the online version of this summary at
General information on gene therapy is available from the Oak Ridge National Laboratory
Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation)
The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis gives details on treatment trials for Leber congenital amaurosis
The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud)
The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis
Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews
WonderBaby, information for parents of babies with Leber congenital amaurosis
PMCID: PMC1896221  PMID: 17594175
10.  In vivo imaging of retinal pigment epithelium cells in age related macular degeneration 
Biomedical Optics Express  2013;4(11):2527-2539.
Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan’s method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm2, which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.
PMCID: PMC3829547  PMID: 24298413
(110.1080) Active or adaptive optics; (330.5310) Vision - photoreceptors; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
11.  Light-induced photoreceptor and RPE degeneration involve zinc toxicity and are attenuated by pyruvate, nicotinamide, or cyclic light 
Molecular Vision  2010;16:2639-2652.
Light-induced damage can be a problem after surgery or sun exposure. Short-duration, intense light causes preferential photoreceptor death in the superior central retina of albino mice and rats and serves as a model of oxidation-induced neurodegeneration. Previous work on retinal ischemia-induced neuronal death suggests the involvement of zinc (Zn2+) toxicity in the death and collapse of many retinal cell layers and demonstrates the protective efficacy of pyruvate. Retinal pigment epithelial (RPE) cells were shown to be sensitive to oxidative stress, and zinc, causing loss of nicotinamide adenine dinucleotide (NAD+) and adenine triphosphate (ATP), which was prevented by pyruvate and nicotinamide. We previously showed similar results in cortical neurons exposed to oxidative stress or Zn2+. In vivo, Zn2+ is normally present in the inner and outer segments (associated with rhodopsin), Bruch’s membrane and sclera (elastin), RPE, and the outer plexiform layer of the eye (synaptic). In this study, we examine the role of Zn2+ in oxidative stress and light-induced damage in vitro and in vivo.
We modeled retinal toxicity in cell-culture lines derived from retinal tissue: Müller and human retinal pigment epithelial (ARPE-19) cells and a cone photoreceptor-derived line (661W). These cultures were exposed to Zn2+ and OS, and the therapeutic efficacy of pyruvate, nicotinamide, and NAD+ was determined. Sprague Dawley albino rats were exposed to 18 kLux of white fluorescent light for 1–4 h in the presence and absence of pyruvate, nicotinamide, lactate, and cyclic light. The intracellular free zinc concentration ([Zn2+]i) and cell damage were assessed 0.5 and 7 days later, respectively.
We show that Zn2+ and oxidative stress results in increased [Zn2+]i and that Zn2+ therapeutic compounds (pyruvate, nicotinamide, and NAD+) and inhibitors of previously implicated pathways (sirtuin) are efficacious in vitro. Exposure to 18 kLux of cool white fluorescent light for 1 h induced a large increase in Zn2+ staining 4–14 h later, particularly in the superior outer nuclear layer and RPE of dark-maintained Sprague Dawley albino rats; 4 h of light was required to induce similar damage in cyclic light-maintained rats. Photoreceptors and RPE cells died in untreated animals at 3–7 days. However, nicotinamide and pyruvate (intraperitoneal), but not lactate, attenuated this death in treated animals, as measured using optical coherence tomography and confirmed by counting photoreceptor nuclei.
Zn2+ plays a role in this injury, as suggested by the increased Zn2+ staining and the efficacy of Zn2+ therapeutics. These results suggest that cyclic light maintenance, Zn2+ chelation, pyruvate, and nicotinamide promote RPE and photoreceptor survival after injury and could be effective for various forms of retinal neurodegeneration. These results could have immediate clinical applications in surgery- or sun exposure- induced light damage to the retina.
PMCID: PMC3002969  PMID: 21179242
12.  Histopathology and Functional Correlations in a Patient with a Mutation in RPE65, the Gene for Retinol Isomerase 
This analysis describes the histopathologic features in the eyes of an adult donor with an RPE65 mutation. The donor had a full clinical workup from visits through several decades, and details are included as background for the histopathology and immunocytochemical analysis. This is the first time an adult donation with a mutation in the RPE65 gene was ever available for study.
Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51.
Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors.
Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons.
The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.
PMCID: PMC3208160  PMID: 21931134
13.  Optimization of In Vivo Confocal Autofluorescence Imaging of the Ocular Fundus in Mice and Its Application to Models of Human Retinal Degeneration 
Standardized imaging procedures allow quantitative and qualitative assessment of fundus autofluorescence in mice. The technique will be useful as an outcome measure in preclinical trials aimed at lowering RPE-lipofuscin and for correlating findings on fundus autofluorescence with postmortem analysis.
To investigate the feasibility and to identify sources of experimental variability of quantitative and qualitative fundus autofluorescence (AF) assessment in mice.
Blue (488 nm) and near-infrared (790 nm) fundus AF imaging was performed in various mouse strains and disease models (129S2, C57Bl/6, Abca4−/−, C3H-Pde6brd1/rd1, Rho−/−, and BALB/c mice) using a commercially available scanning laser ophthalmoscope. Gray-level analysis was used to explore factors influencing fundus AF measurements.
A contact lens avoided cataract development and resulted in consistent fundus AF recordings. Fundus illumination and magnification were sensitive to changes of the camera position. Standardized adjustment of the recorded confocal plane and consideration of the pupil area allowed reproducible recording of fundus AF from the retinal pigment epithelium with an intersession coefficient of repeatability of ±22%. Photopigment bleaching occurred during the first 1.5 seconds of exposure to 488 nm blue light (∼10 mW/cm2), resulting in an increase of fundus AF. In addition, there was a slight decrease in fundus AF during prolonged blue light exposure. Fundus AF at 488 nm was low in animals with an absence of a normal visual cycle, and high in BALB/c and Abca4−/− mice. Degenerative alterations in Pde6brd1/rd1 and Rho−/− were reminiscent of findings in human retinal disease.
Investigation of retinal phenotypes in mice is possible in vivo using standardized fundus AF imaging. Correlation with postmortem analysis is likely to lead to further understanding of human disease phenotypes and of retinal degenerations in general. Fundus AF imaging may be useful as an outcome measure in preclinical trials, such as for monitoring effects aimed at lowering lipofuscin accumulation in the retinal pigment epithelium.
PMCID: PMC3317405  PMID: 22169101
14.  Toward an Understanding of Bisretinoid Autofluorescence Bleaching and Recovery 
To understand molecular mechanisms underlying photobleaching of the RPE fluorophores responsible for fundus autofluorescence.
ARPE-19 cells were allowed to accumulate the bisretinoid, A2E, and were irradiated at 430 nm. For some experiments, the cells were pretreated with vitamin E or sulforaphane and N-acetylcysteine; samples included A2E-free cells. The cells were analyzed by fluorescence microscopy and ultra–performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. A2E free cells were also irradiated and analyzed. Cell death was quantified by double labeling with a membrane impermeable dye and 4′,6′-diamino-2-phenylindole (DAPI).
A2E that had accumulated in ARPE-19 cells exhibited irradiation-associated autofluorescence bleaching despite the absence of appreciable cell death. Chromatographic analysis with absorbance, fluorescence, and mass spectrometry detection revealed that irradiation of A2E was associated with A2E photoisomerization, photooxidation, and photodegradation. Pretreatment with vitamin E favored fluorescence recovery; this finding was consistent with a process involving photooxidation. A2E that was not cell-associated underwent irradiation-induced bleaching, but fluorescence recovery was not observed.
Using cell-associated A2E as a model of RPE bisretinoid behavior, photobleaching and autofluorescence recovery was observed; these changes were similar to RPE autofluorescence reduction in vivo. The potential for autofluorescence recovery is dependent on light dose and antioxidant status. Fluorescence bleaching of bisretinoid involves photooxidative and photodegradative processes.
Fundus autofluorescence bleaching involves bisretinoid photooxidation and photodegradation, the extent of which is dependent on light dose.
PMCID: PMC3390008  PMID: 22570342
15.  Fundus Autofluorescence Findings in a Mouse Model of Retinal Detachment 
Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD).
RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4–5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis.
In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence.
In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.
Autofluorescent puncta that are a feature of retinal degeneration when imaged by fundus autofluorescence, may reflect retinal folds and/or rosettes within which photoreceptor outer segments form hyperfluorescent cores.
PMCID: PMC3416030  PMID: 22786896
16.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
PMCID: PMC1592340  PMID: 17032058
17.  Quantitative Fundus Autofluorescence in Recessive Stargardt Disease 
To quantify fundus autofluorescence (qAF) in patients with recessive Stargardt disease (STGD1).
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.
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.
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.
Quantitative fundus autofluorescence (qAF) is significantly increased in Stargardt disease, consistent with previous reports of increased RPE lipofuscin. QAF will help to establish genotype-phenotype correlations and may serve as an outcome measure in clinical trials.
PMCID: PMC4008047  PMID: 24677105
ABCA4; lipofuscin; retinal pigment epithelium; scanning laser ophthalmoscope; quantitative fundus autofluorescence; recessive Stargardt disease
18.  Age-Dependent Retinal Iron Accumulation and Degeneration in Hepcidin Knockout Mice 
Hepcidin is an iron regulatory hormone expressed in the retina. In the present study, evidence from mice and tissue culture suggest that hepcidin is upregulated in response to increased retinal iron levels and normally serves to prevent retinal iron excess.
Iron dysregulation can cause retinal disease, yet retinal iron regulatory mechanisms are incompletely understood. The peptide hormone hepcidin (Hepc) limits iron uptake from the intestine by triggering degradation of the iron transporter ferroportin (Fpn). Given that Hepc is expressed in the retina and Fpn is expressed in cells constituting the blood-retinal barrier, the authors tested whether the retina may produce Hepc to limit retinal iron import.
Retinas of Hepc−/− mice were analyzed by histology, autofluorescence spectral analysis, atomic absorption spectrophotometry, Perls' iron stain, and immunofluorescence to assess iron-handling proteins. Retinal Hepc mRNA was evaluated through qPCR after intravitreal iron injection. Mechanisms of retinal Hepc upregulation were tested by Western blot analysis. A retinal capillary endothelial cell culture system was used to assess the effect of exogenous Hepc on Fpn.
Hepc−/− mice experienced age-dependent increases in retinal iron followed by retinal degeneration with autofluorescent RPE, photoreceptor death, and subretinal neovascularization. Hepc−/− mice had increased Fpn immunoreactivity in vascular endothelial cells. Conversely, in cultured retinal capillary endothelial cells, exogenous Hepc decreased both Fpn levels and iron transport. The retina can sense increased iron levels, upregulating Hepc after phosphorylation of extracellular signal regulated kinases.
These findings indicate that Hepc is essential for retinal iron regulation. In the absence of Hepc, retinal degeneration occurs. Increases in Hepc mRNA levels after intravitreal iron injection combined with Hepc-mediated decreases in iron export from cultured retinal capillary endothelial cells suggest that the retina may use Hepc for its tissue-specific iron regulation.
PMCID: PMC3053271  PMID: 20811044
19.  Autofluorescence imaging after selective RPE laser treatment in macular diseases and clinical outcome: a pilot study 
The British Journal of Ophthalmology  2002;86(10):1099-1106.
Aim: Selective retinal pigment epithelium (RPE) laser treatment is a new technique which selectively damages the RPE while sparing the neural retina. One difficulty is the inability to visualise the laser lesions. The aim of the study was to investigate whether fundus autofluorescence (AF) is changed because of the RPE damage, and thus might be used for treatment control. Additionally, the clinical course of patients with various macular diseases was evaluated.
Methods: 26 patients with macular diseases (diabetic maculopathy (DMP), soft drusen maculopathy (AMD), and central serous retinopathy (CSR)) were treated and followed up for at least 6 months. Treatment was performed with a train of repetitive short laser pulses (800 ns) of a frequency doubled Nd:YAG laser (parameters: 532 nm, 50 and 500 pulses at 100 and 500 Hz, retinal spot diameter 200 μm, pulse energies 75–175 μJ). AF was excited by 488 nm and detected by a barrier filter at 500 nm (HRA, Heidelberg Engineering, Germany). Patients were examined by ophthalmoscopy, fluorescein angiography, and autofluorescence measurements at various times after treatment (10 minutes, 1 hour, 1 and 6 weeks, 3, 6, and 12 months).
Results: Fluorescein angiography showed leakage from the irradiated areas for about 1 week after treatment. None of the laser lesions was ophthalmoscopically visible during treatment. Identification of the lesions was possible by AF imaging showing an intensity decay in the irradiated area in 22 out of 26 patients, predominantly in patients with CSR and AMD. Lesions could be identified 10 minutes after treatment as hypoautofluorescent spots, which were more pronounced 1 hour later. During follow up the laser spots became hyperautofluorescent. In patients with DMP some AF images were less helpful because of diffuse oedema and larger retinal thickness. In these cases ICG angiography was able to confirm therapeutic success very well. Most of the patients have had benefit from the treatment, with best results obtained for CSR patients.
Conclusion: Imaging of non-visible selective RPE laser effects can be achieved by AF measurements predominantly in patients without retinal oedema. Therefore, AF may replace invasive fluorescein angiography in many cases to verify therapeutic laser success. Selective laser treatment has the potential to improve the prognosis of macular diseases without the risk of laser scotomas.
PMCID: PMC1771314  PMID: 12234886
retinal pigment epithelium; laser photocoagulation; microphotocoagulation; autofluorescence; diabetic maculopathy; drusen; central serous retinopathy
20.  Epiretinal membrane surgery for combined hamartoma of the retina and retinal pigment epithelium: role of multimodal analysis 
The purpose of this study was to evaluate the role of spectral domain optical coherence tomography (SD-OCT), MP-1 microperimetry, and fundus autofluorescence imaging for planning surgical procedures in combined hamartomas of the retina and retinal pigment epithelium (CHR-RPE) and following epiretinal membrane removal.
In an interventional retrospective case series, six consecutive subjects with CHR-RPE underwent vitrectomy and epiretinal membrane peeling, with 4 years of follow-up. Each underwent complete ophthalmic examination, including best corrected visual acuity, fundus examination, fundus fluorescein angiography, SD-OCT, MP-1, and fundus autofluorescence at one, 6, 12, and 48 months.
Six eyes from six subjects with CHR-RPE were studied (mean age 31 ± 14 years). All patients were phakic and five were male (83.3%). Lesions were unilateral, ie, three macular, two juxtapapillary and macular, and one pericentral. Preoperative best corrected visual acuity was 0.3 ± 0.08 Snellen, with significant improvement to 0.9 ± 0.17 Snellen (P = 0.001) at 4 years of follow-up. Mean retinal sensitivity within the central 20° field improved from 16.6 ± 1.84 dB to 18.8 ± 0.96 dB (P = 0.07). There was also a statistically significant reduction in the visual defect (P = 0.04). SD-OCT demonstrated that the epiretinal membranes were completely removed in all but one patient, with significantly decreased macular edema on follow-up at one, 6, 12, and 48 months (P = 0.001). A positive correlation was shown between preoperative macular sensitivity and postoperative best corrected visual acuity. Fundus autofluorescence demonstrated a block in background autofluorescence at the site of the lesion, and hyperautofluorescence at the edematous retina overlain by the epiretinal membrane.
Surgery is an effective treatment for CHR-RPE. SD-OCT, fundus autofluorescence, and MP-1 are valuable and noninvasive tools to guide surgical procedures for CHR-RPE. To the best of our knowledge, this study represents the first use of MP-1 in CHR-RPE in conjunction with SD-OCT and fundus autofluorescence imaging for better guided surgery as well as anatomical and functional prognosis.
PMCID: PMC3553654  PMID: 23378735
vitrectomy; epiretinal membrane; combined hamartoma of the retina and retinal pigment epithelium
21.  Analysis of the RPE sheet in the rd10 retinal degeneration model 
The normal RPE sheet in the C57BL/6J mouse is subclassified into two major tiling patterns: a regular generally hexagonal array covering most of the surface and a “soft network” near the ciliary body made of irregularly shaped cells. Physics models predict these two patterns based on contractility and elasticity of the RPE cell, and strength of cellular adhesion between cells.
We hypothesized and identified major changes in RPE regular hexagonal tiling pattern in rd10 compared to C57BL/6J mice.
In rd10 mice, RPE sheet damage was extensive but occurred later than expected, after most retinal degeneration was complete. RPE sheet changes occur in zones with a bullseye pattern. In the posterior zone, around the optic nerve, RPE cells take on larger irregular and varied shapes to maintain an intact monolayer. In mid periphery, RPE cells have a compressed or convoluted morphology that progress into ingrown layers of RPE under the retina. Cells in the periphery maintain their shape and size until the late stages of the RPE reorganization. The number of neighboring cells varies widely depending on zone and progression. RPE morphology continues to deteriorate after the photoreceptors have degenerated.
The RPE cells are bystanders to photoreceptor degeneration in the rd10 model, and the collateral damage to the RPE results in changes in morphology as early as 30 days old. Quantitative measures of the tiling patterns and histopathology detected here were scripted in a pipeline written in Perl and Cell Profiler (an open source MatLab plugin) and are directly applicable to RPE sheet images from noninvasive fundus autofluorescence (FAF), adaptive optics confocal scanning laser ophthalmoscope (AO-cSLO), and spectral domain optical coherence tomography (SD-OCT) of patients with early stage AMD or RP.
PMCID: PMC3732179  PMID: 22183388
22.  Retinal Pigment Epithelium Defects in Humans and Mice with Mutations in MYO7A: Imaging Melanosome-Specific Autofluorescence 
Usher syndrome (USH) is a genetically heterogeneous disease with autosomal recessive deafness and blindness. Gene therapy is under development for use in the most common genetic variant of USH1, USH1B, which is caused by mutations in the MYO7A gene. This study was undertaken to identify an imaging method for noninvasively monitoring the RPE component of the USH1B disease.
NIR-autofluorescence (NIR-AF) was examined in USH1B patients with scanning laser ophthalmoscopy, and retinal thickness with spectral-domain optical coherence tomography. Myo7a-null mouse retinas and purified RPE melanosomes were analyzed by spectral deconvolution confocal microscopy.
In USH1B patients, NIR-AF was normal in regions of retained photoreceptors and abnormal in regions lacking photoreceptors. Subtle changes in NIR-AF were associated with intermediate photoreceptor loss. In ex vivo mouse retinas, the NIR-AF source was traced to the melanosomes in the RPE and choroid. Purified RPE melanosomes emitted the same signal. Fluorophores, excited by long-wavelength light, were evident throughout the apical RPE of WT mouse eyecups. In Myo7a-null eyecups, these fluorophores had a more restricted distribution. They were absent from the apical processes of the RPE, thus correlating with the melanosome localization defects described previously by conventional microscopy.
The data indicate that melanosomes in the RPE and choroid are the dominant source of NIR-AF from the posterior region of the eye. NIR-AF is a novel tool that provides sensitive and label-free imaging of the retina and RPE and is currently the only melanosome-specific, noninvasive technique for monitoring RPE disease in new therapeutic initiatives for retinal degenerations.
PMCID: PMC2884175  PMID: 19324852
23.  The Oral Iron Chelator Deferiprone Protects Against Systemic Iron Overload–Induced Retinal Degeneration in Hepcidin Knockout Mice 
To investigate the retinal-protective effects of the oral iron chelator deferiprone (DFP) in mice lacking the iron regulatory hormone hepcidin (Hepc). These Hepc knockout (KO) mice have age-dependent systemic and retinal iron accumulation leading to retinal degeneration.
Hepc KO mice were given DFP in drinking water from age 6 to 18 months. They were then compared to Hepc KO mice not receiving DFP by fundus imaging, electroretinography (ERG), histology, immunofluorescence, and quantitative PCR to investigate the protective effect of DFP against retinal and retinal pigment epithelial (RPE) degeneration.
In Hepc KO mice, DFP diminished RPE depigmentation and autofluorescence on fundus imaging. Autofluorescence in the RPE layer in cryosections was significantly diminished by DFP, consistent with the fundus images. Immunolabeling with L-ferritin and transferrin receptor antibodies showed a decreased signal for L-ferritin in the inner retina and RPE cells and an increased signal for transferrin receptor in the inner retina, indicating diminished retinal iron levels with DFP treatment. Plastic sections showed that photoreceptor and RPE cells were well preserved in Hepc KO mice treated with DFP. Consistent with photoreceptor protection, the mRNA level of rhodopsin was significantly higher in retinas treated with DFP. The mRNA levels of oxidative stress–related genes heme oxygenase-1 and catalase were significantly lower in DFP-treated Hepc KO retinas. Finally, ERG rod a- and b- and cone b-wave amplitudes were significantly higher in DFP-treated mice.
Long-term treatment with the oral iron chelator DFP diminished retinal and RPE iron levels and oxidative stress, providing significant protection against retinal degeneration caused by chronic systemic iron overload in Hepc KO mice. This indicates that iron chelation could be a long-term preventive treatment for retinal disease involving iron overload and oxidative stress.
Iron chelation provides a long-term preventive treatment for retinal disease involving iron overload and oxidative stress.
PMCID: PMC4106252  PMID: 24970260
deferiprone; oxidative stress; hepcidin; retinal degeneration
24.  Autofluorescence Imaging for Diagnosis and Follow-up of Cystoid Macular Edema 
Lipofuscin results from digestion of photoreceptor outer segments by the retinal pigment epithelium (RPE) and is the principal compound that causes RPE fluorescence during autofluorescence imaging. Absorption of the 488-nanometer blue light by macular pigments, especially by the carotenoids lutein and zeaxanthin, causes normal macular hypo-autofluorescence. Fundus autofluorescence imaging is being increasingly employed in ophthalmic practice to diagnose and monitor patients with a variety of retinal disorders. In macular edema for example, areas of hyper-autofluorescence are usually present which are postulated to be due to dispersion of macular pigments by pockets of intraretinal fluid. For this reason, the masking effect of macular pigments is reduced and the natural autofluorescence of lipofuscin can be observed without interference. In cystic types of macular edema, e.g. cystoid macular edema due to retinal vein occlusion, diabetic macular edema and post cataract surgery, hyper-autofluorescent regions corresponding to cystic spaces of fluid accumulation can be identified. In addition, the amount of hyper-autofluorescence seems to correspond to the severity of edema. Hence, autofluorescence imaging, as a noninvasive technique, can provide valuable information on cystoid macular edema in terms of diagnosis, follow-up and efficacy of treatment.
PMCID: PMC3520597  PMID: 23264870
Autofluorescence; Cystoid Macular Edema; Lipofuscin
25.  The drusen-like phenotype in aging Ccl2 knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages 
Drusen, which can be defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2 knockout (Ccl2-/-) mice have been reported to develop drusen and phenotypic features similar to AMD including an increased susceptibility to choroidal neovascularisation (CNV). Here we investigate the nature of the drusen-like lesions in vivo and further evaluate the Ccl2-/- mouse as a model for AMD.
We examined eyes of 2-25 month old Ccl2-/- and C57Bl/6 mice in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO), electroretinography, and measured the extent of laser- induced CNV by fluorescein fundus angiography. We also assessed retinal morphology using immunohistochemistry and quantitative histological and ultrastructural morphometry.
The drusen-like lesions of Ccl2-/- mice comprise accelerated accumulation of swollen CD68+, F4/80+ macrophages in the subretinal space that are apparent as autofluorescent foci on AF-SLO. These macrophages contain pigment granules and phagosomes with outer segment and lipofuscin inclusions that might account for their autofluorescence. We only observed age-related RPE damage, photoreceptor loss and sub-RPE deposits but, despite the accelerated accumulation of macrophages, we identified no spontaneous CNV development in senescent mice and found a reduced susceptibility to laser-induced CNV in Ccl2-/- mice.
These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2-/- mice that are similar to AMD may be the result of aging alone.
PMCID: PMC2801148  PMID: 19578022
Aging; MCP-1/Ccl2 knockout mouse; subretinal macrophages; Laser-induced CNV; autofluorescent SLO imaging; age-related macular degeneration; AMD; HRA2

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