Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects.

PURPOSE: To characterize the age-related accumulation of lipofuscin in a population of normal subjects, resolve differences in estimated accumulation rates obtained in previous studies, and characterize the spatial distribution of lipofuscin in the normal fundus. METHODS: Spectrophotometric measurements were made at the fovea and 7 degrees temporal to the fovea in 145 normal subjects (age range, 15-80 years). Spatial distribution along the four cardinal meridians was measured in selected subjects by both spectrophotometry and autofluorescence imaging. To minimize contributions of extraneous fluorophores, macular pigment, and melanin, all measurements used excitation at 550 nm, integrating emission between 650 and 750 nm. RESULTS: Lipofuscin fluorescence increased linearly until age 70, then declined. The rate of accumulation was significantly slower in the fovea than at the temporal site; accumulation rates in vivo were greater than previously observed in microscopic studies. Fluorescence was approximately 40% lower in the fovea than at 7 degrees eccentricity and was asymmetrically distributed around the fovea. The fluorescence was maximal at approximately 11 degrees temporally, approximately 7 degrees nasally, approximately 13 degrees superiorly, and approximately 9 degrees inferiorly. At the same eccentricity, fluorescence was always less along the inferior meridian than along any other. CONCLUSIONS: Light absorption by RPE melanin can explain differences between the in vivo and ex vivo estimates of the rate of lipofuscin accumulation. Declining fluorescence at old age may represent removal of atrophic RPE cells. The spatial distribution of lipofuscin generally matches that of rods and reflects, rather than predicts, the pattern of age-related loss of rod photoreceptors.

Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry.

We present a technique for estimating the density of the human macular pigment noninvasively that takes advantage of the autofluorescence of lipofuscin, which is normally present in the human retinal pigment epithelium. By measuring the intensity of fluorescence at 710 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, we can make accurate single-pass measurements of the macular pigment density. We used the technique to measure macular pigment density in a group of 159 subjects with normal retinal status ranging in age between 15 and 80 years. Average macular pigment density was 0.48 +/- 0.16 density unit (D.U.) for a 2 degrees -diameter test field. We show that these estimates are highly correlated with reflectometric (mean: 0.23 +/- 0.07 D.U.) and psychophysical (mean: 0.37 +/- 0.26 D.U.; obtained by heterochromatic flicker photometry) estimates of macular pigment in the same subjects, despite the fact that systematic differences in the estimated density exist between techniques. Repeat measurements over both short- and long-time intervals indicate that the autofluorescence technique is reproducible: The mean absolute difference between estimates was less than 0.05 D.U., superior to the reproducibility obtained by reflectometry and flicker photometry. To understand the systematic differences between density estimates obtained from the different methods, we analyzed the underlying assumptions of each technique. Specifically, we looked at the effect of self-screening by visual pigment, the effect of changes in optical property of the deeper retinal layers, including the role of retinal pigmented epithelium melanin, and the role of secondary fluorophores and reflectors in the anterior layers of the retina.

Photodynamic therapy using Lu-Tex induces apoptosis in vitro, and its effect is potentiated by angiostatin in retinal capillary endothelial cells.

PURPOSE: To examine the effect of combining angiostatin with photodynamic therapy (PDT) using Lutetium Texaphyrin (Lu-Tex; Alcon, Fort Worth, TX) as a photosensitizer in bovine retinal capillary endothelial (BRCE) and retinal pigment epithelial (RPE) cells and to determine the mode of PDT-induced cell death in these cell lines. METHODS: Cultured BRCE and RPE cells were incubated with angiostatin (500 ng/ml) for 18 hours and subjected to Lu-Tex/PDT, using treatment parameters previously optimized (3 microgram/ml Lu-Tex for 30 minutes followed by timed irradiation at 732 nm). Cellular survival was assessed after a 1-week cellular proliferation. Data were analyzed using Student's t-test. Caspase 3 activity was monitored in cells after PDT using a fluorogenic substrate, (Asp-Glu-Val-Asp)-AFC (7-amino-4-trifluoromethyl coumarin) [DEVD-AFC], of caspase 3. After PDT, expression of Bcl-2, Bcl-x(L), Bax, and Bak was also examined in cell lysates by Western blot analysis. RESULTS: A synergistic cytotoxic effect of angiostatin and Lu-Tex/PDT was observed in BRCE cells at all fluences used (5, 10, and 20 J/cm(2); P

Autofluorescence distribution associated with drusen in age-related macular degeneration.

PURPOSE: To determine whether drusen in patients with age-related maculopathy and macular degeneration (ARM/AMD) are associated with focal changes in retinal pigment epithelium (RPE) lipofuscin fluorescence. METHOD: A new autofluorescence imaging device was used to study lipofuscin distribution associated with individual drusen in 20 patients with ARM/AMD. Paired monochromatic and autofluorescence fundus images were used for detailed analysis of the topography of autofluorescence at specific sites containing drusen. In four eyes, image analysis was used to compare the spatial distribution of the autofluorescence with the location of drusen and to quantify the autofluorescence distribution over individual drusen (54 drusen). REsuLTs. A specific pattern of autofluorescence was frequently found to be spatially associated with hard drusen and soft drusen between 60 and 175 microm in size. The pattern is characterized by a central area of decreased autofluorescence surrounded, in most cases, by an annulus of increased autofluorescence. The location of this pattern was highly correlated with the position of individual distinct drusen. The central low autofluorescence focus was on average 16% below the surrounding background, and the annulus, when present, was on average 6% more fluorescent than the background. Soft drusen larger than 175 microm and confluent soft drusen show either multifocal areas of low autofluorescence or a more heterogeneous distribution. CONCLUSIoNs. Autofluorescence imaging permits measurement of RPE lipofuscin at specific sites. RPE overlying drusen have altered autofluorescence, suggesting changes in RPE health.

Diffusion of high molecular weight compounds through sclera.

PURPOSE: To determine the in vitro permeability of the sclera to high molecular weight compounds and the relationship between scleral permeability and molecular size. METHODS: Fresh rabbit sclera was mounted in a two-chamber diffusion apparatus, and its permeability to sodium fluorescein, fluorescein isothiocyanate (FITC)-conjugated bovine serum albumin, FITC-IgG, and FITC dextrans ranging in molecular weight from 4 to 150 kDa was determined by fluorescence spectrophotometry. Electron microscopy was used to assess the impact of the experimental design on scleral ultrastructural integrity. The effect of the diffusion apparatus on scleral hydration was examined. Rabbit scleral permeability was compared with previously reported data for human and bovine sclera. RESULTS: Scleral permeability decreased with increasing molecular weight and molecular radius, consistent with previous human and bovine data. Molecular radius was a better predictor of scleral permeability than molecular weight. The sclera was more permeable to globular proteins than to linear dextrans of similar molecular weight. The experimental apparatus did not alter scleral ultrastructure. Permeability of rabbit sclera was similar to human sclera but greater than bovine sclera. CONCLUSIONS: Large molecules, such as IgG, diffuse across sclera in a manner consistent with porous diffusion through a fiber matrix. Transscleral delivery of immunoglobulins and other large compounds to the choroid and retina may be feasible.

Transscleral delivery of bioactive protein to the choroid and retina.

PURPOSE: To investigate the feasibility of transscleral drug delivery to the choroid and retina. METHODS: An osmotic pump was used to deliver IgG across the sclera of pigmented rabbits, and levels were measured in the choroid, retina, vitreous humor, aqueous humor, orbit, and plasma over 28 days. This method was then used to deliver an anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (mAb), and its effect on inhibiting vascular endothelial growth factor (VEGF)-induced leukostasis in the choroid and retina was determined by measuring tissue myeloperoxidase (MPO) activity. RESULTS: Levels of retinal and choroidal IgG were significantly higher than baseline at all points up to 28 days (P < or = 0.01). IgG levels in the orbit, vitreous humor, aqueous humor, and plasma were negligible (P > 0.05). MPO activity in the choroid of eyes treated with anti-ICAM-1 mAb was 80% less (P = 0.01) than in eyes receiving an equal rate of delivery of an isotype control antibody. Inhibition of MPO activity in the retina was 70% (P = 0.01). The plasma concentration of anti-ICAM-1 mAb was 31,000-fold less than the concentration in the osmotic pump. CONCLUSIONS: Minimally invasive transscleral delivery can be used to deliver therapeutic levels of bioactive drugs to the choroid and retina with negligible systemic absorption. This method of ocular drug delivery may be used in the treatment of a variety of chorioretinal disorders.

Reflectance and curvature of the inner limiting membrane at the foveola.

Light reflected specularly by the inner limiting membrane (ILM) provides information on the topography of the retinal surface. The ILM in the central part of the foveal pit acts as a concave mirror. Light reflected specularly by this mirror forms an image of the entrance pupil in front of the retina. In 15 normal subjects (ages 16-56 years) we have measured photometric and geometrical-properties of this image to derive two characteristics of the ILM: its reflectance rho at the foveola and its radius of curvature r in the central part of the fovea. rho and r are found to decrease significantly with age (p = 0.0073 and p = 0.01, respectively). The equations of the regression lines are log10 rho = -4.234 - 0.0118 age and radius r = 1484 - 13.6 age, respectively (age in years, r in micrometers).

Fundus reflectance and the measurement of crystalline lens density.

We have developed an optical technique for measuring the in vivo absorption of the human crystalline lens based on using the retina as a reflector for a double-pass measurement of lens density. We compare results obtained by the reflectometric technique with results obtained in the same subjects by a psychophysical method. We find that the estimates obtained with the two techniques are highly correlated, and both measure similar effects of aging on the optical density of the lens. In addition, we show that the significant variations in retinal reflectance attributable to differences in ocular pigmentation and drusen have only a minor effect on the estimate of lens optical density. However, correcting retinal reflectances for the effect of lens absorption shows that the effect of lens absorption may be overestimated with both methods. The overestimation may arise from inaccuracies in the shape of the assumed lens spectrum.

Infrared imaging of sub-retinal structures in the human ocular fundus.

The interaction of infrared light with the human ocular fundus, particularly sub-retinal structures, was studied in vivo. Visible and infra-red wavelengths and a scanning laser ophthalmoscope were used to acquire digital images of the human fundus. The contrast and reflectance of selected retinal and sub-retinal features were computed for a series of wavelengths or modes of imaging. Near infrared light provides better visibility than visible light for sub-retinal features. Sub-retinal deposits appear light and thickened; the optic nerve head, retinal vessels, and choroidal vessels appear dark. Contrast and visibility of features increases with increasing wavelength from 795 to 895 nm. Optimizing the mode of imaging improves the visibility of some structures. This new quantitative basis for near infrared imaging techniques can be applied to a wide range of imaging modalities for the study of pathophysiology and treatment in diseases affecting the retinal pigment epithelium and Bruch's membrane, such as age-related macular degeneration.

In vivo measurement of lipofuscin in Stargardt's disease--Fundus flavimaculatus.

PURPOSE: Several histopathologic studies have concluded that Stargardt's disease (Fundus flavimaculatus) is associated with abnormally high levels of lipofuscin-like material in the retinal pigment epithelium. The purpose of this study was to determine whether this material has the same fluorescence characteristics as lipofuscin in vivo and whether noninvasive measurements identify a significant elevation in this material. METHODS: Five patients with autosomal recessive Stargardt's disease were included in this study, as were 45 healthy controls. All patients had the angiographic dark choroid sign. The intensity and emission spectra of lipofuscin fluorescence were measured by noninvasive fundus spectrophotometry at 7 degrees temporal to the fovea. RESULTS: The fluorescence intensities in the five patients with Stargardt's disease were significantly higher (P < 0.0001) than those observed in normal subjects of the same age. The emission spectra in the patients are similar in shape to those measured in normals, but flecks appear to shift the spectra toward shorter wavelengths. CONCLUSIONS: The spectral characteristics of the fluorophore observed in patients with Stargardt's disease are consistent with those of retinal pigment epithelial lipofuscin. These patients have abnormally high levels of lipofuscin, confirming previous histopathologic observations. Noninvasive retinal pigment epithelial lipofuscin measurements may be a useful adjunct in the diagnosis of Stargardt's disease.-F. flavimaculatus.

[In vivo fundus fluorescence measurements in patients with age related macular degeneration]. / In-vivo-Fundus-Fluoreszenzmessungen bei Patienten mit altersabhängiger Makuladegeneration.

UNLABELLED: This study was performed to measure and characterize the intrinsic fluorescence of the ocular fundus in patients with age-related macular degeneration (AMD). METHOD: Fluorescence spectral measurements from discrete retinal locations were made using the fundus spectrophotometer with excitations at 470 and 510 nm. Two normal subjects and seven patients with different stages of AMD were investigated. RESULTS: The spectral characteristics of fundus fluorescence are consistent with those of lipofuscin in the retinal pigment epithelium (RPE). The fluorescence spectrum is broad, with a maximum at about 620 nm. The shape and intensity of the fluorescence spectra are affected by age, site of measurement, pathology, ocular media absorption, and excitation wavelength. Spectra from areas with drusen reveal an additional fluorophore, with maximum around 560 nm, probably emanating from drusen and Bruch's membrane. Measurements in atrophic reveal a decrease of lipofuscin fluorescence and/or a contribution likely due to choroidal and sclera collagen fluorescence. Fluorescence from lipofuscin is more efficiently excited at 510 nm, whereas that of drusen and subretinal structures is relatively more efficient with 470 nm excitation, allowing for discrimination of various fluorophores. CONCLUSION: The spectral characteristics of RPE lipofuscin could be identified and quantified in AMD patients. In addition, the spectra are affected by other fluorophores such as drusen and choroid contributions in atrophy. Fluorescence spectra measurements in AMD patients allow for discrimination of lipofuscin fluorescence, drusen fluorescence, and choroidal or scleral fluorescence. The non-invasive measurement of lipofuscin and drusen fluorescence in AMD may be helpful in monitoring the disease, understanding its evolution, and testing therapeutic concepts.

Comparison of fluorescein angiography with microvascular anatomy of macaque retinas.

Recent anatomic work has shown that the capillary network of the fovea is multilaminar. We have identified the elements of this network that are visualized by fluorescein angiography and those that are missed. Fluorescein angiograms of monkey retinas (Macaca fascicularis) with good visualization of individual capillaries were obtained by standard clinical techniques. Retinal whole mounts were prepared from the same animals. Anatomic drawings made from the whole mounts were used to identify which parts of the capillary network were visualized angiographically. Angiographic estimates of dimensions of the foveal avascular zone corresponded closely to the anatomy. Capillary visibility declined rapidly from near perfect visualization at the edge of the foveal avascular zone to less than 40% by 900 microns eccentricity. While all the widest capillary segments (diameter 6.1-7.0 microns) were visualized, only 43% of the modal group of capillary segments (diameter 4.1-4.5 microns) were detected. When a relatively homogeneous population of capillaries was analyzed (diameters limited to the narrow range of 4.0-5.0 microns), visualization declined monotonically with depth in the retina. Capillary segments in the nerve fiber plane were visualized more than four times as effectively as segments of comparable diameter in the deepest vascular plane. High quality angiograms accurately delineate the foveal avascular zone, but they visualize only a fraction of the adjacent multilaminar network. Therefore, current techniques may not detect the earliest nonperfusion of capillaries in vaso-occlusive diseases. Capillary visibility is a joint function of diameter and of retinal depth. The decline in visualization with retinal depth implies that light scattering in the retina degrades the angiographic image.

In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics.

PURPOSE: To characterize the intrinsic fluorescence (autofluorescence) of the human ocular fundus with regard to its excitation and emission spectra, age relationship, retinal location, and topography, and to identify the dominant fluorophore among the fundus layers. METHODS: Using a novel fundus spectrophotometer, fluorescence measurements were made at 7 degrees temporal to the fovea and at the fovea in 30 normal subjects and in 3 selected patients. Topographic measurements were made in 3 subjects. Ex vivo measurements of fluorescence of human retinal pigment epithelium (RPE) were obtained and compared to in vivo data. RESULTS: Fundus fluorescence reveals a broad band of emission between 500 and 750 nm, a maximum of approximately 630 nm, and optimal excitation of approximately 510 nm. Exhibiting a significant increase with age, this fluorescence is highest at 7 degrees to 15 degrees from the fovea, shows a well-defined foveal minimum, and decreases toward the periphery. In vivo fluorescence spectra are consistent with those obtained ex vivo on human RPE. Measurements with short wavelength excitation are strongly influenced by ocular media absorption and reveal an additional minor fluorophore in the fovea. CONCLUSIONS: Spectral characteristics, correlation with age, topographic distribution, and retinal location between the choriocapillaris and the photoreceptors suggest that the dominant fundus fluorophore is RPE lipofuscin. The minor fluorophore is probably in the neurosensory retina but has not been identified.

Spectrophotometer for noninvasive measurement of intrinsic fluorescence and reflectance of the ocular fundus.

A spectrophotometer for noninvasively measuring the intrinsic fluorescence and the reflectance of the ocular fundus is described. The instrument uses multichannel spectral analysis for recording fluorescence emission spectra (500-800 nm) with seven excitation wavelengths between 430 and 550 nm and for the determination of fundus reflectance spectra (400-800 nm). Measurements are performed from a discrete fundus area, with a spatial resolution of a 1-2° visual angle. Calibration procedures are detailed. Representative fluorescence and reflectance spectra obtained from five normal subjects indicate that the fluorescence originates from within the fundus layers. Although the absolute fundus fluorescence measurement is affected by lens absorption and ocular refraction, it is minimally influenced by the strong fluorescence of the crystalline lens.

Growth of cultured RPE and endothelial cells is inhibited by blue light but not green or red light.

Blue light, but not green or red light, inhibited growth of retinal pigment epithelial (RPE) cells, aortic endothelial cells, and fibroblasts in vitro. Significant inhibition was observed in all 3 cell types exposed for 18 hr to blue light (425-500 nm) at 42 J/cm2. Damage was prevented by inclusion of superoxide dismutase (SOD) and catalase, providing evidence for a photooxidative mechanism. Dopa (100 microM) also caused oxidative damage that suppressed growth of all 3 cell types. A synergism of dopa and light effects was observed in endothelial cells and fibroblasts, but the agents caused additive effects on RPE cells. Endothelial cells were the most sensitive to dopa, light, and the two combined. Fibroblasts were the only cell type that exhibited greater sensitivity to light than to dopa. These data suggest that oxygen-mediated damage to the growing blood vessels in the retina of a premature infant may be exacerbated by exposure to blue light. A further implication is that restriction of RPE melanogenesis to the prenatal period of darkness and lower oxygen protects the retina from simultaneous oxidative challenge by light and by reactive species generated during oxidation of dopa released to the extracellular environment.

Effects of optic atrophy on retinal blood flow and oxygen saturation in humans.

Retinal blood flow and oxygen saturation were evaluated in patients with unilateral inner retinal degeneration secondary to neurogenic optic atrophy. Arteriovenous O2 saturation for temporal and nasal vascular segments of the affected eyes, evaluated by retinal vessel oximetry, was 12% +/- 9% higher than in the fellow eyes (seven patients). Blood flow in the temporal retinal arteries of the affected eyes, measured by the laser Doppler technique, was 48% +/- 20% lower than in the fellow eyes (four patients). The combination of these results indicated a 40% +/- 29% reduction in O2 delivery in the affected eyes (four patients), thereby quantifying the decrease in retinal metabolism that resulted from inner retinal degeneration.

Spectral reflectance of the human ocular fundus.

Reflectance spectra from discrete sites in the human ocular fundus were measured with an experimental reflectometer in the visible and near-infrared parts of the spectrum. The principal study population consisted of ten subjects 22 to 38 years of age with a wide range of degree of fundus melanin pigmentation. Reflectance spectra were obtained from the nasal fundus, the fovea, and an area 2.5 degrees from the fovea. Spectra were also recorded from several older subjects and from one aphakic patient with a coloboma. The reflectance spectra were found to be influenced by the degree of individual and local melanin pigmentation of the fundus, the amount of blood in the choroid, the transmission properties of the ocular media, and the discrete reflections in the stratified fundus layers. Mathematical models of the optical properties of the stratified layers are proposed and are fitted to the experimental fundus reflectance spectra. The models account for the absorption by blood, melanin, macular pigment, and ocular media, and incorporate tissue scattering and discrete reflectors corresponding to anatomical layers.

Fundus reflectometry for photocoagulation dosimetry.

A dosimetry technique has been developed which utilizes three-wavelength fundus reflectometry, a quantitative model of fundus reflectance, and a model of thermal tissue damage to control photocoagulation lesion size. The fundus reflectance model uses Lambert-Beer's law exponential attenuators to describe the ocular media, retinal pigment epithelium (RPE), and choriocapillaris transmission characteristics while the choroid is described as a Kubelka-Munklike homogeneous scatterer/absorber. Three reflecting layers are included in the model at the retinal inner limiting membrane, Bruch's membrane, and the sclera. Measured lesion size variability contained components which resulted from variations in choroidal blood and melanin and RPE melanin concentration. Photocoagulation dosimetry was found to reduce the photocoagulation lesion size coefficient of variation for red light from 45% to 10% for the control and dosimetry cases, respectively. Similar improvement was noted for yellow photocoagulation light.

Evaluation of micrometric and microdensitometric methods for measuring the width of retinal vessel images on fundus photographs.

The width of retinal vessel images on fundus photographs was determined by projection micrometry and microdensitometry. These methods were evaluated on 12 retinal vessels using the negatives of 570-nm monochromatic fundus photographs. For projection micrometry, the intraobserver reproducibility of vessel width measurements was 1.6%-2.9%, depending upon the experience of the observer. Significant interobserver differences in the measured widths were demonstrated. For microdensitometry, three distinct measurement criteria were used. Significant differences in width as measured by the three criteria were found, but there were no interoperator differences for each criterion. The intraoperator reproducibility of vessel width measurements by microdensitometry was 2.1%-2.5%. Significant differences were found in the vessel widths determined by the micrometry and densitometry methods, and results obtained by micrometry are discussed in terms of edge-detection phenomena.