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.
We have previously shown that long exposure to 568 nm light at levels below the maximum permissible exposure safety limit produces retinal damage preceded by a transient reduction in the autofluorescence of retinal pigment epithelial (RPE) cells in vivo. Here, we determine how the effects of exposure power and duration combine to produce this autofluorescence reduction and find the minimum exposure causing a detectable autofluorescence reduction.
Macaque retinas were imaged using a fluorescence adaptive optics scanning laser ophthalmoscope to resolve individual RPE cells in vivo. The retina was exposed to 568 nm light over a square subtending 0.5° with energies ranging from 1 J/cm2 to 788 J/cm2, where power and duration were independently varied.
In vivo exposures of 5 J/cm2 and higher caused an immediate decrease in autofluorescence followed by either full autofluorescence recovery (exposures ≤ 210 J/cm2) or permanent RPE cell damage (exposures ≥ 247 J/cm2). No significant autofluorescence reduction was observed for exposures of 2 J/cm2 and lower. Reciprocity of exposure power and duration held for the exposures tested, implying that the total energy delivered to the retina, rather than its distribution in time, determines the amount of autofluorescence reduction.
That reciprocity holds is consistent with a photochemical origin, which may or may not cause retinal degeneration. The implementation of safe methods for delivering light to the retina requires a better understanding of the mechanism causing autofluorescence reduction. Finally, RPE imaging was demonstrated using light levels that do not cause a detectable reduction in autofluorescence.
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.
A2E; all-trans-retinal; bisretinoid; retinal pigment epithelium; macular degeneration; retina
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.
Autofluorescence; Cystoid Macular Edema; Lipofuscin
Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified
confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in
front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy
disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE
cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell
mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees
of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for
improving the visualization of the RPE cell boundaries including: pinhole diameter, filament
thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus,
wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The
demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light
exposures relative to light safety standards and it is more comfortable for the subject than the
traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field
imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening
and monitoring disease progression.
(170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering; (110.1080) Active or adaptive optics
To develop a clinical planning module (CPM) to improve the efficiency of imaging subjects with a steerable wide-field adaptive optics scanning laser ophthalmoscope (AOSLO). To evaluate the performance of this module by imaging the retina in healthy and diseased eyes.
We developed a software-based CPM with two sub-modules: a navigation module and a montage acquisition module. The navigation module guides the AOSLO to image identified retinal regions from a clinical imaging platform using a matrix-based mapping between the two. The montage acquisition module systematically moves the AOSLO steering mirrors across the retina in predefined patterns. The CPM was calibrated using a model eye and tested on five normal subjects and one patient with a retinal nerve fiber layer (RNFL) defect.
Within the central +/−7 degrees from the fixation target, the CPM can direct the AOSLO beam to the desired regions with localization errors of less than 0.3 degrees. The navigation error increases with eccentricity, and larger errors (up to 0.8 degrees) were evident for regions beyond 7 degrees. The repeatability of CPM navigation was tested on the same locations from 2 subjects. The localization errors between trials on different days did not differ significantly (p >0.05). The region with a size of approximately 13 by 10 degrees can be imaged in about 30minutes. An approximately 12 × 4.5 degree montage of the diseased region from a patient was imaged in 18 minutes.
We have implemented a clinical planning module to accurately guide the AOSLO imaging beam to desired locations, and to quickly acquire high resolution AOSLO montages. The approach is not only friendly for patients and clinicians, but also convenient to relate the imaging data between different imaging platforms.
adaptive optics; scanning laser ophthalmoscope; retinal nerve fiber layer; clinical planning module
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.
Phototoxicity; Photochemical; Retina; Retinal pigment epithelium; Autofluorescence; Visual cycle; Lipofuscin; Bisretinoids
Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor αvβ5 for particle binding. Here, we tested RPE phagocytosis and retinal function in β5 integrin–deficient mice, which specifically lack αvβ5 receptors. Retinal photoresponses severely declined with age in β5−/− mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. β5−/− RPE in culture failed to take up isolated photoreceptor particles. β5−/− RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in β5−/− retina. These results demonstrate an essential role for αvβ5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the β5−/− integrin mouse strain as a new animal model of age-related retinal dysfunction.
circadian rhythm; knockout; photoreceptors; retinal pigment; epithelium; vision
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution.
The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes.
(110.1080) Active or adaptive optics; (330.5380) Physiology; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
Adaptive optics scanning laser ophthalmoscopy demonstrated abnormal cone spacing in regions of abnormal fundus autofluorescence and reduced visual function in 12 patients with Stargardt disease.
To study the relationship between macular cone structure, fundus autofluorescence (AF), and visual function in patients with Stargardt disease (STGD).
High-resolution images of the macula were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography in 12 patients with STGD and 27 age-matched healthy subjects. Measures of retinal structure and AF were correlated with visual function, including best-corrected visual acuity, color vision, kinetic and static perimetry, fundus-guided microperimetry, and full-field electroretinography. Mutation analysis of the ABCA4 gene was completed in all patients.
Patients were 15 to 55 years old, and visual acuity ranged from 20/25–20/320. Central scotomas were present in all patients, although the fovea was spared in three patients. The earliest cone spacing abnormalities were observed in regions of homogeneous AF, normal visual function, and normal outer retinal structure. Outer retinal structure and AF were most normal near the optic disc. Longitudinal studies showed progressive increases in AF followed by reduced AF associated with losses of visual sensitivity, outer retinal layers, and cones. At least one disease-causing mutation in the ABCA4 gene was identified in 11 of 12 patients studied; 1 of 12 patients showed no disease-causing ABCA4 mutations.
AOSLO imaging demonstrated abnormal cone spacing in regions of abnormal fundus AF and reduced visual function. These findings provide support for a model of disease progression in which lipofuscin accumulation results in homogeneously increased AF with cone spacing abnormalities, followed by heterogeneously increased AF with cone loss, then reduced AF with cone and RPE cell death. (ClinicalTrials.gov number, NCT00254605.)
Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood–retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side – that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.
neurodegeneration; photoreceptor; VEGF; retina; gene therapy
Chorioretinal folds typically involve the choroid, Bruch membrane, retinal pigment epithelium (RPE), and sometimes overlying neurosensory retina. von Winning hypothesized that the alternate banding pattern of choroidal folds shown by fluorescein angiography is explained by RPE density. To our knowledge, autofluorescence imaging of chorioretinal folds has not been previously described.
A 47-year-old healthy hyperopic man had best-corrected visual acuity of 20/30 in the right eye and 20/25 in the left eye. Posterior segment examination revealed bilateral chorioretinal folds with subtle streaks of RPE hyperpigmentation and hypopigmentation emanating from both optic nerve heads.
Early-phase fluorescein angiography revealed the characteristic pattern of alternating light and dark bands. Autofluorescence imaging disclosed a similar pattern as well as peripapillary mottling. The alternating patterns of light and dark bands observed using autofluorescence imaging and fluorescein angiography were found to be precisely in register but inverted.
Autofluorescence imaging noninvasively demonstrates the pathognomonic pattern of alternating light and dark bands shown by fluorescein angiography diagnostic of choroidal folds but in an inverse fashion. This observation provides independent support of von Winning’s hypothesis regarding the etiopathogenesis of the banding pattern.
chorioretinal folds; autofluorescence; fluorescein angiography; hyperopia; retinal pigment epithelium
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.
vitrectomy; epiretinal membrane; combined hamartoma of the retina and retinal pigment epithelium
To establish adaptive optics scanning laser ophthalmoscopy (AOSLO) as a method to detect and characterize microscopic signs of diabetic retinopathy, in capillaries and cone photoreceptors in the parafovea.
Recently, AOSLO has enabled noninvasive assessment of photoreceptors, capillaries, and leukocytes in the retinas of live human subjects. Repeated application of AOSLO imaging along with comparison to fluorescein angiography was used to track individual capillaries near the foveal avascular zone (FAZ) from one eye affected with severe non-proliferative diabetic retinopathy. Fluorescein angiography was used to identify clinical signs of diabetic retinopathy, such as microaneurysms and intra-retinal microvascular abnormalities (IRMAs), and corresponding regions were imaged and assessed using the AOSLO. In addition, the structural integrity of photoreceptors and the spatial distribution of leukocytes around the parafoveal capillary network were quantitatively assessed.
Capillaries and cone photoreceptors were visualized using the AOSLO, without the use of injected contrast agents. Although the majority of capillaries were stable over a period of 16 months, one capillary at the edge of the FAZ dropped out, leading to a small, but significant increase in FAZ size. Longitudinal assessment of the capillaries also showed microaneurysm formation and disappearance, as well as the formation of tiny capillary bends similar in appearance to IRMAs. The leukocytes in the capillary network were found to preferentially travel through the same routes in all four visits, suggesting that these channels are robust against small changes to the surrounding capillaries. In this eye, cone photoreceptor spacing was increased in the fovea when compared to normal data, but stable across all visits.
AOSLO imaging can be used to longitudinally track capillaries, leukocytes and photoreceptors in diabetic retinopathy. Capillary changes that can be detected include dropout of individual capillaries, as well as formation and disappearance of microaneurysms.
adaptive optics; capillaries; diabetic retinopathy; leukocyte; microaneurysm; photoreceptors
To investigate the retinal microvasculature at the fovea and peripheral retina in humans using the adaptive optics scanning laser ophthalmoscope (AOSLO). To examine the association of foveal avascular zone (FAZ) and foveal pit morphology.
Retinal imaging of the foveal capillary network was performed on 11 subjects (15 eyes; age range 20–54) with an AOSLO. Standard deviation maps of the AOSLO images were generated from ~10–30 frames, producing high resolution maps delineating the complete capillary distribution of the retina. Foveal pit morphology was investigated in the same subjects by using a spectral domain optical coherence tomography (SDOCT). In an additional subject, only a relatively large retinal vasculature map was obtained using AOSLO.
A well demarcated FAZ was seen in 11 subjects tested with foveal capillary imaging. There was considerable individual variation in the size and shape of the FAZ. The mean FAZ area and mean FAZ effective diameter were 0.33mm2 and 622μm, respectively. Foveal thickness was found to be negatively correlated with the FAZ effective diameter.
The structure of the capillary network could be evaluated in the fovea and parafovea using our approach. We find that a smaller FAZ is associated with a narrower foveal pit opening and a thicker fovea.
foveal avascular zone; foveal pit morphology; adaptive optics scanning laser ophthalmoscope; spectral domain optical coherence tomography; retina; retinal thickness
We have developed a new, unified implementation of the adaptive optics scanning laser ophthalmoscope (AOSLO) incorporating a wide-field line-scanning ophthalmoscope (LSO) and a closed-loop optical retinal tracker. AOSLO raster scans are deflected by the integrated tracking mirrors so that direct AOSLO stabilization is automatic during tracking. The wide-field imager and large-spherical-mirror optical interface design, as well as a large-stroke deformable mirror (DM), enable the AOSLO image field to be corrected at any retinal coordinates of interest in a field of >25 deg. AO performance was assessed by imaging individuals with a range of refractive errors. In most subjects, image contrast was measurable at spatial frequencies close to the diffraction limit. Closed-loop optical (hardware) tracking performance was assessed by comparing sequential image series with and without stabilization. Though usually better than 10 μm rms, or 0.03 deg, tracking does not yet stabilize to single cone precision but significantly improves average image quality and increases the number of frames that can be successfully aligned by software-based post-processing methods. The new optical interface allows the high-resolution imaging field to be placed anywhere within the wide field without requiring the subject to re-fixate, enabling easier retinal navigation and faster, more efficient AOSLO montage capture and stitching.
The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.
The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency.
The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.
To study tissue remodelling and wound healing after retinal pigment epithelium (RPE) tears due to age-related macular degeneration.
Retrospective longitudinal study of 36 eyes (33 patients) with RPE tears. Imaging was performed using fundus autofluorescence (FAF) (λ=488 nm) and spectral-domain optical coherence tomography (SD-OCT). Presence of intraretinal hyper-reflective dots in SD-OCT, which correlated with hyperfluorescent dots in FAF, indicating RPE migration was studied. Morphology of subretinal mass and RPE layer integrity in the RPE denuded area over time were examined.
7 of 36 eyes (19.4%) showed patchy or hazy hyperfluorescent areas in FAF, and the majority of eyes (83.3%) showed hyper-reflective dots, which possibly represent intraretinal RPE migration and hard exudates. Homogenous subretinal mass was encountered in about half of all cases. In one case (2.8%), the RPE layer proliferated and covered the defect.
SD-OCT and FAF showed a considerable amount of RPE proliferation, migration and repopulation. Intraretinal RPE migration did not form a functional RPE layer. A small defect might be repaired by cell proliferation. But this RPE proliferation is not sufficient to cover large defects.
Retinal pigment epithelium tear; pigment migration; spectral-domain optical coherence tomography; fundus autofluorescence; macula; pharmacology; neovascularisation; drugs; clinical trial; treatment surgery
This is the first in vivo study of cone photoreceptor packing density at different ages.
To study the variation of cone photoreceptor packing density across the retina in healthy subjects of different ages.
High-resolution adaptive optics scanning laser ophthalmoscope (AOSLO) systems were used to systematically image the retinas of two groups of subjects of different ages. Ten younger subjects (age range, 22–35 years) and 10 older subjects (age range, 50–65 years) were tested. Strips of cone photoreceptors, approximately 12° × 1.8° long were imaged for each of the four primary retinal meridians: superior, inferior, nasal, and temporal. Cone photoreceptors within the strips were counted, and cone photoreceptor packing density was calculated. Statistical analysis (three-way ANOVA) was used to calculate the interaction for cone photoreceptor packing density between age, meridian, and eccentricity.
As expected, cone photoreceptor packing density was higher close to the fovea and decreased with increasing retinal eccentricity from 0.18 to 3.5 mm (∼0.6–12°). Older subjects had approximately 75% of the cone density at 0.18 mm (∼0.6°), and this difference decreased rapidly with eccentricity, with the two groups having similar cone photoreceptor packing densities beyond 0.5 mm retinal eccentricity on average.
Cone packing density in the living human retina decreases as a function of age within the foveal center with the largest difference being found at our most central measurement site. At all ages, the retina showed meridional difference in cone densities, with cone photoreceptor packing density decreasing faster with increasing eccentricity in the vertical dimensions than in the horizontal dimensions.
To describe the retinal imaging findings in the index patient with Heimler syndrome (OMIM #234580).
Non-interventional case report.
A 29-year-old woman with Heimler syndrome developed bilateral vision loss. Fluorescein angiography (FA), fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT) and electroretinography (ERG) were performed to assess the retinal anatomy and function.
FA showed mottling of the retinal pigment epithelium (RPE) in the posterior pole and periphery of the retina. FAF revealed hyper and hypoautofluorescent dots corresponding to the RPE mottling observed on FA. SD-OCT documented loss of the inner/outer segments boundary, and RPE thinning. ERG testing excluded generalized rod-cone dysfunction.
We report an adult-onset macular dystrophy in one of the previously reported patients with Heimler syndrome and hypothesize that this syndrome is probably an expression of a ciliopathy.
Amelogenesis imperfecta; Ciliopathy; Deafness; Heimler syndrome; Nail abnormalities; Retina
To correlate the degree of functional loss with structural changes in patients with Stargardt disease.
Eighteen eyes of 10 Stargardt patients were studied. Scanning laser ophthalmoscope (SLO) infrared images were compared to corresponding spectral domain optical coherence tomography (SD-OCT) scans. Additionally, SLO microperimetry was performed and results were superimposed on SLO infrared images and in selected cases on fundus autofluorescence (FAF) images.
Seventeen of 18 eyes showed a distinct hypo-reflective foveal and/or perifoveal area with distinct borders on SLO-infrared images which was less evident on funduscopy and incompletely depicted in FAF images. This hypo-reflective zone corresponded to areas of significantly elevated psychophysical thresholds on microperimetry testing, in addition to thinning of the retinal pigment epithelium (RPE), disorganization or loss of the photoreceptor cell inner-outer segment (IS-OS) junction and external limiting membrane (ELM) on SD-OCT.
SLO-infrared fundus images are useful for depicting retinal structural changes in Stargardt patients. An SD-OCT/SLO microperimetry device allows for a direct correlation of structural abnormalities with functional defects that will likely be applicable for the determination of retinal areas for potential improvement of retinal function in these patients during future clinical trials and for the monitoring of the diseases' natural history.
microperimetry; SLO infrared imaging; Stargardt disease; fundus autofluorescence imaging
Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.
To establish the characteristics of secondary retinal and retinal pigment epithelial (RPE) changes associated with the presence of choroidal melanoma and choroidal nevus as documented by optical coherence tomography (OCT) and fundus autofluorescence (FAF).
Materials and Methods:
PubMed review of major English publications examining the correlation between clinical characteristics of choroidal melanoma and nevus with OCT and FAF findings.
The intrinsic properties of choroidal melanoma, as well as overlying RPE changes, drusen, and lipofuscin are best characterized by FAF, while OCT is more sensitive for the identification of subretinal and intraretinal fluid as well as atrophy, degeneration, and photoreceptor loss in the neurosensory retina.
Secondary retinal changes associated with choroidal melanocytic lesions can be documented by OCT and FAF. OCT-evident changes are observed more often with choroidal melanoma than choroidal nevus. OCT is better suited to identify the overlying retinal detachment and edema, even before these findings are clinically apparent. FAF is most useful in documenting the presence of lipofuscin, a finding that represents one of the important criteria in differentiating small choroidal melanoma from benign choroidal nevus.
Autofluorescence; Choroid; Eye; Melanoma; Nevus; Optical Coherence Tomography
To ascertain the potential pathogenicity of a retinitis pigmentosa (RP)-causing RHO F45L allele in a family affected by congenital achromatopsia (ACHM).
Case series/observational study that included two patients with ACHM and 24 extended family members. Molecular genetic analysis was performed to identify RHO F45L carrier status in the family and a control population. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Comprehensive clinical testing included acuity, color vision, and dilated fundus examination. Electroretinography was used to assess rod and cone function.
Five carriers of the RHO F45L allele alone (24–80 years) and three carriers in combination with a heterozygous CNGA3 mutant allele (10–64 years) were all free of the classic symptoms and signs of RP. In heterozygous carriers of both mutations, SD-OCT showed normal retinal thickness and intact outer retinal layers; rod and cone densities were within normal limits on AOSLO. The phenotype in two individuals affected with ACHM and harboring the RHO F45L allele was indistinguishable from that previously reported for ACHM.
The RHO F45L allele is not pathogenic in this large family; hence, the two ACHM patients would unlikely develop RP in the future.
The combined approach of comprehensive molecular analysis of individual genomes and noninvasive cellular resolution retinal imaging enhances the current repertoire of clinical diagnostic tools, giving a substantial impetus to personalized medicine.
exome sequencing; adaptive optics; rhodopsin mutations; retinitis pigmentosa; retinal degeneration