Visualization of retinal pigment epithelial cells in vivo using digital high-resolution confocal scanning laser ophthalmoscopy

doi:10.1016/j.ajo.2003.08.004

American Journal of Ophthalmology

Volume 137, Issue 3, March 2004, Pages 556-558

https://doi.org/10.1016/j.ajo.2003.08.004 Get rights and content

Abstract

Purpose

To visualize retinal pigment epithelial cells in vivo by fundus autofluorescence imaging using a confocal scanning laser ophthalmoscope.

Design

Experimental study and observational case report.

Methods

Digital in vivo autofluorescence images were recorded with a confocal scanning laser ophthalmoscope (excitation, 488 nm; emission, >500 nm) and compared with confocal scanning laser ophthalmoscope and fluorescence microscopic recordings from human donor eyes.

Results

A uniform pattern of the polygonal retinal pigment epithelial cell layer was visualized in vivo outside of absorbing retinal vessels and macular pigment. Autofluorescence intensities of individual cells showed marked variation. The pattern corresponded to in vitro findings. Visualization is based on the topographic distribution of autofluorescent lipofuscin granules and melanin granules in apical retinal pigment epithelium cytoplasm.

Conclusions

High-resolution autofluorescence imaging may be useful to determine morphologic and lipofuscin-dependent alterations in retinal diseases and may be applicable for monitoring effects of therapeutic interventions targeting the retinal pigment epithelium.

References (5)

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Distribution of pigment epithelium autofluorescence in retinal disease state recorded in vivo and its change over time
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F.G. Holz et al.
Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration
Invest Ophthalmol Vis Sci
(2001)

There are more references available in the full text version of this article.

Cited by (36)

Correlation between fundus autofluorescence and central visual function in chronic central serous chorioretinopathy
2015, American Journal of Ophthalmology
To find possible correlations between the morphologic macular changes revealed by fundus autofluorescence (FAF) and the functional parameters such as visual acuity and retinal sensitivity in patients with chronic central serous chorioretinopathy (CSC).
Prospective, cross-sectional study.
Forty-six eyes (39 consecutive patients) with chronic CSC were studied with FAF and microperimetry (MP). Retinal sensitivity value maps were exactly superimposed over FAF images. The following microperimetric parameters were applied: central 10-degree visual field, 4-2-1 strategy, 61 stimulation spots, white monochromatic background, stimulation time 200 ms, stimulation spot size Goldmann III. A possible relationship between MP and FAF was investigated.
Mean best-corrected visual acuity (BCVA) was 20/32 (median 20/25, range 20/20–20/200). BCVA was significantly correlated with FAF findings (Mann-Whitney test; P < .0001). A positive concordance between FAF and MP evaluation was also found (total concordance of 0.720 with a kappa of Cohen of 0.456). The hypo-autofluorescent areas showed decreased retinal sensitivity, while adjacent areas of increased FAF could be associated to both normal and decreased retinal sensitivity. Absolute scotoma, defined as 0 dB retinal sensitivity, corresponded with absence of autofluorescence.
Altered FAF in chronic CSC patients has a functional correlation quantified by microperimetry. This study confirms the impact of FAF changes on retinal sensitivity and their value to reflect the functional impairment in chronic CSC.
Peripheral autofluorescence and clinical findings in neovascular and non-neovascular age-related macular degeneration
2013, Ophthalmology
Citation Excerpt :
Earlier studies on the FAF features of AMD have focused on the central 30 degrees,18,19,23,24 partly because of limitations of the equipment and the inability to image the periphery in a convenient noncontact manner. A variety of central FAF patterns, particularly in the junctional zones surrounding atrophic lesions, have been described in detail,8,18,19,23 and some patterns (e.g., banded or diffuse junctional FAF) were identified to be of potential prognostic value. In the longitudinal natural history arm of the Fundus Autofluorescence in age-related Macular degeneration (FAM) study,8 classification of eyes according to FAF pattern at baseline seemed to have stronger predictive value for progression of atrophy over time compared with other risk factors, including baseline size of geographic atrophy.
To characterize peripheral fundus autofluorescence (FAF) abnormalities in patients with age-related macular degeneration (AMD), correlate these with clinical findings, and identify risk factors associated with these FAF abnormalities.
Clinic-based, cross-sectional study.
A total of 119 consecutive patients: 100 patients with AMD (200 eyes) and 19 patients without AMD (38 eyes).
In a prospective study performed at the Doheny Eye Institute, University of Southern California, widefield 200-degree FAF and color images were obtained by the Optos 200Tx Ultra-Widefield device (Optos, Dunfermline, Scotland) using a standardized imaging protocol. The FAF images were captured centered on the fovea, and additional images were captured after steering the field of view inferiorly and superiorly. All FAF and color images were graded independently by 2 masked ophthalmologists with respect to the presence, location, extent, and type of peripheral (defined as outside the central 30 degrees) FAF abnormality.
Presence and type of peripheral FAF abnormalities.
Peripheral FAF abnormalities were evident in 164 eyes (68.9%), with several distinct FAF patterns identified: granular (46.2%), mottled (34.0%), and nummular (18.1%). A 90% concordance of FAF patterns was observed between both eyes. Abnormal FAF occurred more frequently in neovascular compared with non-neovascular AMD or normal eyes (86% vs. 72.8% vs. 18.4%, respectively, P<0.001). Significant risk factors for peripheral FAF abnormalities were AMD type (neovascular AMD odds ratio [OR], 12.7 and non-neovascular AMD OR, 6.2 compared with normal eyes, P<0.001), older age (OR, 6.5; 95% confidence interval [CI], 2.4–17.8; P<0.001 for the oldest quartile compared with the youngest), and female sex (OR, 4.1; 95% CI, 1.9–8.9; P<0.001). Clinical features on color photography were detected in 174 eyes (73.1%): peripheral drusen (51.7%), retinal pigment epithelium (RPE) depigmentation (34.9%), RPE hyperpigmentation (branching reticular pigmentation) (22.7%), and atrophic patches (16.8%). There was a high correlation between specific FAF and clinical findings: granular FAF with peripheral drusen (P<0.001) and mottled FAF with RPE depigmentation (P<0.001).
Several distinct patterns of peripheral FAF abnormalities were observed in 68.9% of patients, with AMD type, female sex, and age being independent risk factors. The peripheral FAF patterns correlate strongly with specific clinical features seen in eyes with AMD.
Proprietary or commercial disclosure may be found after the references.
Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells
2010, Experimental Eye Research
Citation Excerpt :
We performed emission scans at 380 nm excitation and excitation scans at 570 nm emission to allow for comparability with previous studies (Sparrow et al., 1999; Wassell et al., 1998). In addition, we employed a 488 nm excitation wavelength that correlates with the excitation wavelength used in our fluorescence microscopy and flow cytometry studies as well as in clinical application of RPE autofluorescence detection (Bindewald et al., 2004). Consistent with our flow cytometry results, overall cellular autofluorescence intensities measured by spectroscopy were increased after incubation with HNE- and MDA-modified POS compared to unmodified POS (Fig. 3).
Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.
Progression of Geographic Atrophy and Impact of Fundus Autofluorescence Patterns in Age-related Macular Degeneration
2007, American Journal of Ophthalmology
Citation Excerpt :
In a combined analysis of the FAF pattern classification and the classification of baseline total atrophy, there was a trend of a smaller GA size at baseline within the group with no abnormal and focal FAF pattern, but statistical analysis could not abandon the thesis of the independence between both classification systems (P = .070; Table 3). With the advent of digital confocal scanning laser ophthalmoscopy, it has become possible to visualize LF accumulation at the level of the RPE cell monolayer in vivo.19–21,35 FAF imaging gives additional information above and beyond conventional imaging tools such as fundus photography, fluorescein angiography, or optical coherence tomography.
To test if fundus autofluorescence (FAF) patterns around geographic atrophy (GA) have an impact on GA progression rates over time in atrophic age-related macular degeneration (AMD).
Prospective longitudinal multicenter natural history study.
Standardized digital FAF images were obtained from 195 eyes of 129 patients with GA using confocal scanning laser ophthalmoscopy (excitation 488 nm, emission >500 nm). Areas of GA were quantified and patterns of abnormal FAF in the junctional zone were classified. Repeated FAF images were obtained over a median follow-up period of 1.80 years (interquartile range [IQR], 1.28 to 3.34).
Areas of GA (median, 7.04 mm² at baseline; IQR, 3.12 to 10.0) showed a median enlargement of 1.52 mm²/year (IQR, 0.81 to 2.33). Progression rates in eyes with the banded (median 1.81 mm²/year) and the diffuse FAF pattern (1.77 mm²/year) were significantly higher compared to eyes without FAF abnormalities (0.38 mm²/year) and focal FAF patterns (0.81 mm²/year, P < .0001). Within the group of the diffuse pattern, eyes with a diffuse trickling pattern could be identified that exhibited an even higher spread rate (median 3.02 mm²/year) compared to the other diffuse types (1.67 mm²/year, P = .001).
The results indicate that distinct phenotypic FAF patterns have an impact on disease progression in eyes with atrophic AMD and may therefore serve as prognostic determinants. The findings underscore the relevance of FAF imaging and the pathogenetic role of excessive retinal pigment epithelium (RPE) lipofuscin (LF) accumulation in GA. Natural history data and identification of high-risk characteristics will be helpful to design interventional studies aiming at slowing the spread of atrophy.
Behavioral and anatomical abnormalities in a sodium iodate-induced model of retinal pigment epithelium degeneration
2006, Experimental Eye Research
We characterized changes in the visual behavior of mice in which a loss of the retinal pigment epithelium (RPE) was experimentally induced with intravenous (i.v.) administration of sodium iodate (NaIO₃). We compared and correlated these changes with alterations in neural retinal structure and function. RPE loss was induced in 4–6 week old male C57BL/6 mice with an i.v. injection of 1% NaIO₃ at three concentrations: 35, 50, or 70 mg/kg. At 1, 3, 7, 14, 21, and 28 days (d) as well as 6 months post injection (PI) a behavioral test was performed in previously trained mice to evaluate visual function. Eye morphology was then assessed for changes in both the RPE and neural retina.
NaIO₃-induced RPE degeneration was both dose and PI time dependent. Our low dose showed no effects, while our high dose caused the most damage, as did longer PI times at our intermediate dose. Using the intermediate dose, no changes were detectable in either visual behavior or retinal morphology at 1 d PI. However, at 3 d PI visual behavior became abnormal and patchy RPE cell loss was observed. From 7 d PI onward, changes in retinal morphology and visual behavior became more severe. At 6 months PI, no recovery was seen in any of these measures in mice administered the intermediate dose. These results show that NaIO₃ dosage and/or time PI can be varied to produce different, yet permanent deficits in retinal morphology and visual function.
Thus, this approach should provide a unique system in which the onset and severity of RPE damage, and its consequences can be manipulated. As such, it should be useful in the assessment of rescue or mitigating effects of retinal or stem cell transplantation on visual function.
The segmentation of zones with increased autofluorescence in the junctional zone of parapapillary atrophy
2009, Physiological Measurement

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Brief reportVisualization of retinal pigment epithelial cells in vivo using digital high-resolution confocal scanning laser ophthalmoscopy

Abstract

Purpose

Design

Methods

Results

Conclusions

Distribution of pigment epithelium autofluorescence in retinal disease state recorded in vivo and its change over time

Graefes Arch Clin Exp Ophthalmol

Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration

Invest Ophthalmol Vis Sci

Brief report
Visualization of retinal pigment epithelial cells in vivo using digital high-resolution confocal scanning laser ophthalmoscopy