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.

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.

Diversity of response of optic nerve head circulation to timolol maleate in gel-forming solution.

PURPOSE: This randomized, double-masked, placebo-controlled, two-period crossover study was conducted to investigate the effects of 0.5% timolol maleate in gel-forming solution on intraocular pressure (IOP) and blood circulation in the optic nerve head in patients with untreated ocular hypertension. METHODS: The effects of 0.5% timolol in gel-forming solution on IOP and optic nerve head capillary blood speed were studied in 12 patients with untreated ocular hypertension. Optic nerve capillary blood speed was measured using the laser Doppler technique before and at the end of each treatment period. RESULTS: In each patient, IOP decreased after treatment with timolol (mean decrease 16.8% versus placebo). Systemic blood pressure and pulse rate did not differ significantly after treatment with topical timolol from values after placebo. The mean change from baseline in Doppler broadening was 10.6% greater after treatment with timolol than after placebo. There was no significant change in mean Doppler broadening from baseline after treatment with either timolol or placebo. However, optic nerve head capillary blood speed increased in six patients, and was within the range of placebo response in six patients after treatment with timolol. Spearman correlation analysis of the baseline with Doppler broadening measurements after treatment showed a correlation for placebo but not for timolol. The percent change in Doppler broadening after timolol treatment was correlated with iris color. CONCLUSION: These results indicate that administration of timolol for 4 weeks reduces IOP in patients with ocular hypertension and generally does not change the blood circulation in the optic nerve head. Individual patients, however, showed variable changes in optic nerve head circulation after topical administration of timolol. Although the sample size was small, these changes in optic nerve head circulation were correlated with iris color.

Optic nerve head circulation after topical calcium channel blocker.

PURPOSE: Our purpose was to investigate the effects of the calcium channel blocker verapamil on intraocular pressure and blood circulation in the human optic nerve head. METHODS: The effects of three different concentrations of topical verapamil (0.063%, 0.125%, and 0.25%) on intraocular pressure and optic nerve head capillary blood speed were measured in 12 healthy normal subjects. In a randomized, double-masked design, each subject received one drop of either verapamil or placebo in one eye and the opposite treatment in the fellow eye. Anterior optic nerve circulation was assessed at baseline and 90 min after instillation of the drops using the laser Doppler technique. RESULTS: The intraocular pressure was significantly reduced compared with baseline in both verapamil- and placebo-treated eyes at each concentration. The reductions of intraocular pressure were greater in verapamil-treated eyes (12-17%) than in placebo-treated eyes (9-12%). No systemic effect on heart rate or blood pressure was detected after administration of topical verapamil. The capillary blood speed in the optic nerve head was increased in both verapamil- and placebo-treated eyes at each concentration, although the only statistically significant increases were with the 0.25% concentration. The mean +/- SEM increase compared with baseline at the 0.25% concentration was 10.4 +/- 3.6% in verapamil-treated eyes (p = 0.017), and 11.6 +/- 4.4% in placebo-treated eyes (p = 0.026). CONCLUSIONS: These results indicate that topical administration of verapamil lowers the intraocular pressure and increases the capillary blood speed in the optic nerve head in normal subjects. Changes measured in verapamil-treated eyes were also observed in placebo-treated eyes, indicating a crossover effect.

Retinal blood flow changes in type I diabetes. A long-term follow-up study.

PURPOSE: The authors previously reported that blood speeds in the retinal arteries were significantly lower in patients with type I diabetes than in controls without diabetes. The purpose of this long-term, follow-up study was to characterize the natural course of changes in blood speed and blood flow in these patients. METHODS: Twenty-four patients were followed up with serial annual measurements of the blood flow in a temporal retinal artery using the bidirectional laser Doppler technique and monochromatic photography. The follow-up period ranged from 2 to 6 years (mean, 3.8 years). Using standardized color fundus photography and fluorescein angiography, a retinopathy score was generated for each eye studied. Linear regression analysis was used to compute the slope of the change in retinal blood flow for each patient during the follow-up period. RESULTS: Retinal blood flow slopes were negative in 15 patients and positive in 9 patients. Multiple linear regression analysis showed that the retinal blood flow slopes were significantly related to the retinal blood flow measured at entry to the study and to the median duration of diabetes during the follow-up period (R2 = 0.56; P = 0.0002). There was a positive correlation between the retinal blood flow slopes and the median retinopathy score during the follow-up period (P = 0.47; P = 0.02). CONCLUSIONS: As duration of diabetes becomes longer and retinopathy becomes more severe, there is a transition from negative to positive retinal blood flow slopes. This bimodal relationship between the change in retinal blood flow and the duration of diabetes reflects the complex pathologic alterations that occur in the diabetic retina.

Retinal blood flow increases following short-term aspirin usage in type I diabetics with no or minimal retinopathy.

In a double-masked, randomized, placebo-controlled, two-period crossover study, we measured the effect of aspirin on retinal blood flow in 8 type I diabetic patients, 5 with no observable retinopathy, and 3 with 1-10 observable microaneurysms and no other signs of retinopathy. Each patient ingested 1 capsule/day of either aspirin (650 mg) or placebo for 14 days. Following a 1-month washout period, the treatment was reversed. The bidirectional laser Doppler technique and monochromatic photography were used to measure the blood flow rate in a major temporal retinal artery in 1 eye of each patient at baseline and on the 14th day of each treatment period. Retinal blood flow increased following aspirin in 7 of the 8 patients. Using a standard crossover analysis we found a mean aspirin-placebo treatment difference of 21 +/- 17% (mean +/- 95% CI), which was statistically significant (p = 0.03). Retinal blood flow increased following aspirin by 44 +/- 10% (mean +/- SEM) in the 5 patients with no retinopathy (p = 0.04), but only by 8 +/- 10% (n.s.) in the 3 patients with minimal retinopathy.

Retinal hemodynamics in middle-aged normal subjects.

The laser Doppler technique and monochromatic photography were used to measure the total retinal blood flow, temporal/nasal differences in blood flow and the relationship between blood flow and vessel diameter in 5 healthy subjects, aged 54-58 years. Systemic blood pressure (BP) and intraocular pressure were also measured, and the retinal perfusion pressure was calculated. The measurements were compared to those previously obtained from a younger group of 7 healthy subjects, aged 25-38 years. Total retinal blood flow was 73 +/- 13 microliters/min in the middle-aged subjects and was not significantly different from the value measured in young subjects (80 +/- 12 microliters/min). Retinal perfusion pressure was significantly higher in the older subjects, primarily due to elevated systemic BP. The similarity in total flow between the two groups, even though the retinal perfusion pressures were higher in the older group, is an indication of an increased vascular resistance to flow. The increase may be an aging phenomenon or an indication of a well-functioning autoregulatory capacity in the retinal vasculature of the older subjects.

Optic nerve head circulation in untreated ocular hypertension.

AIMS: The laser Doppler technique was used to compare the capillary blood speed measured at localised sites of the optic nerve head in stable, untreated ocular hypertensive patients with that measured in healthy normal subjects. The stereophotogrammetric technique was also used to measure the retinal nerve fibre layer thickness at the disc margin in the eyes of the patients. METHODS: Doppler broadening measurements were made at superior and inferior temporal disc sites in 18 eyes of 10 ocular hypertensive patients and in 12 eyes of seven age and sex-matched normal subjects. RESULTS: On average, Doppler broadening and, hence, capillary blood speed were significantly higher (p = 0.018) in the patients than in the normal subjects. The largest values of Doppler broadening in the patients were measured at sites adjacent to the thinnest retinal nerve fibre layer. Linear regression analysis showed a significant inverse relation (p = 0.0004) between Doppler broadening and nerve fibre layer thickness in left eyes, and a nearly significant relation (p = 0.06) in right eyes. At temporal sites of the optic nerve head there is a compensatory relation between a thinning nerve fibre layer and a locally increasing blood supply to the optic nerve head. CONCLUSION: Together with previous observations of fluorescein filling defects in similar patients, these results indicate that there is spatial heterogeneity of blood flow in the optic nerve head in stable, untreated ocular hypertensive patients.

[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.

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 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.

Retinal circulatory abnormalities in type 1 diabetes.

PURPOSE: To quantify retinal circulatory abnormalities in patients with type 1 diabetes; to 1 diabetes; to compare blood speed and blood flow in major temporal retinal arteries as well as total retinal arterial cross-section measured in patients to that measured in controls without diabetes; to determine which factors are related to the measured abnormalities within the patient group. METHODS: The laser Doppler technique and monochromatic fundus photography were used to measure retinal circulatory parameters in 39 patients with type 1 diabetes with duration of diabetes between 7 and 20 years and 13 age-matched controls without diabetes. Blood pressure, intraocular pressure, and heart rate were measured in all subjects. Glycosylated hemoglobin was measured in the patients. Retinopathy was assessed using standardized color fundus photography and fluorescein angiography. RESULTS: Total retinal arterial cross-section was, on average, 17% higher (P = 0.007) in the patients than in the controls, and it increased with increasing duration of diabetes (P = 0.006). Arterial blood speed was, on average, 33% lower (P = 0.0001) in the patients than in the controls, and it decreased with increasing duration of diabetes (P = 0.03). CONCLUSIONS: The retinal circulation of patients with type 1 diabetes with no retinopathy or background retinopathy is characterized by dilated major arteries with reduced blood speeds. Dilation of the larger retinal arteries, with the accompanying decrease in vascular resistance to flow in those vessels, appears to counteract an increase in resistance to flow at the level of the smaller retinal vessels.

Regional retinal blood flow reduction following half fundus photocoagulation treatment.

Regional changes in retinal blood flow following inferior and subsequent superior argon laser half fundus photocoagulation treatment were measured in six diabetic patients with advanced retinopathy. Centreline blood velocity, vessel diameter, and blood flow in major inferior and superior temporal retinal arteries were measured at baseline and following each treatment using the bidirectional laser Doppler technique and monochromatic fundus photography. Inferior fundus laser treatment produced a significant blood flow decrease in inferior temporal arteries ranging from 60% to 78%. Blood flow changes in superior temporal arteries were not significant, ranging from -7% to +14%. Subsequent superior fundus laser treatment produced a significant blood flow decrease in superior temporal arteries ranging from 50% to 66%. Subsequent blood flow changes in inferior temporal arteries were not significant, ranging from -19% to +21%. The results indicate that regional laser treatment produces a regional reduction in retinal blood flow, consistent with measurements of preretinal and intraretinal oxygen tension which have indicated increases in oxygen over photocoagulated regions.

Quantitative circulatory measurements in branch retinal vessel occlusion.

We used the laser Doppler technique to quantify retinal circulatory abnormalities in three patients with branch retinal artery occlusion and in two patients with branch retinal vein occlusion, each of whom had shown delayed filling on fluorescein angiography. Blood flow in occluded arteries was 40-50% lower than in non-occluded arteries of the same branching order. In the patients with branch retinal vein occlusion blood flow in arteries supplying the affected regions was 80-90% lower than in arteries supplying the unaffected regions. One vein occlusion patient was measured serially over 18 months. Blood flow increased from 4 to 12 microliters/min in the artery supplying the affected region in this patient. The results demonstrate that retinal circulatory abnormalities can be evaluated quantitatively during the course of retinal vascular occlusive disease, and suggest that the efficacy of therapy can be monitored using our techniques.

[Retinal blood flow changes in eyes with rhegmatogenous retinal detachment and scleral buckling procedures].

Bidirectional laser Doppler technique and monochromatic photography was used to measure the absolute blood flow rate (F) in the major superior or inferior temporal retinal arteries in 3 patients with rhegmatogenous retinal detachment, in 4 patients following unilateral scleral buckling procedures, and in one patient before and after removal of scleral buckling elements. In 2 of the eyes with retinal detachment, F in arteries supplying the detached portion of the retina was approximately 30% lower than in arteries supplying the attached portion. On average, F measured in the arteries supplying the detached portion of the retina in 2 patients was 8.9 +/- 1.3 microliters/min, 67% lower than normal controls. F measured in the arteries supplying the attached portion of the retina in 3 patients was 15.2 +/- 3.9 microliters/min, on average, 43% lower than in normal controls. In 4 patients who had undergone uncomplicated, unilateral scleral buckling procedures, F in an artery of the affected eye was compared with F in the corresponding artery of the fellow eye. F in the artery of the affected eyes was 8.7 +/- 3.2 microliters/min, on average, 59% lower than that of the fellow eyes (p less than 0.05), and 68% lower than in normal controls (p less than 0.0001). Removal of scleral buckling elements in one patient produced a 79% increase in F in the measured artery. Then, F returned to normal levels after removal of scleral buckling elements.

Optic nerve head blood speed as a function of age in normal human subjects.

We used the laser Doppler technique to determine the relation between age and the speed of blood cells moving through the capillaries of the optic nerve head. We studied 22 normal human volunteers ranging in age from 16-76 years. The results were best described by a statistically significant quadratic relationship between capillary blood speed and age. Blood speeds were lowest in the youngest and oldest subjects and highest in subjects between 27 and 35 years old. A two-phase linear model showed a statistically significant 20% decrease in blood speed in volunteers between the ages of 31 and 76. The results were not affected by gender, degree of refractive error, systemic blood pressure, intraocular pressure, cup/disc ratio of the optic nerve head, or by site-to-site differences in the light scattering properties of the optic nerve head tissue. Capillary blood speed was, on average, 15% greater from temporal sites than from nasal sites, corresponding to the equally greater distribution of ganglion cell axons within the same area. The results provide a baseline of normal age-controlled data that can be compared to measurements obtained from patients with disorders of the optic nerve head thought to have a vascular etiology.

Effect of timolol on human retinal, choroidal and optic nerve head circulation.

In a double-masked, randomized, placebo-controlled study, we evaluated the effect of topical timolol maleate 0.5% on the retinal, choroidal, and optic nerve head circulation in 5 healthy volunteer subjects. Changes in the pulsatile component of choroidal blood flow (PCBF) were determined from measurements of the ocular pulse wave. Changes in the retinal arterial blood flow rate (RBF) and optic nerve head capillary blood speed (CBS) were determined by laser Doppler velocimetry and monochromatic photography. In timolol-treated eyes, PCBF decreased by 32 +/- 12% (p = 0.0007). Changes in RBF and CBS were not statistically significant. In the contralateral placebo-treated eyes, PCBF decreased by 15 +/- 8% (p = 0.006) and RBF increased by 18 +/- 10% (p = 0.002). The change in CBS was not statistically significant.

Blood flow in the normal human retina.

The laser Doppler technique was used to measure the blood flow rate in 41 major vessels in ten eyes of healthy volunteer subjects. The specific relationship between blood flow rate, F, and vessel diameter, D, was determined for both retinal arteries and retinal veins. On average, F increased with increasing D at a power of 4.1, consistent with the presence of Poiseuille flow. In six eyes of six subjects, measurements on individual vessels were combined to yield the total retinal blood flow rate. The mean and standard deviation of the total retinal blood flow was 80 +/- 12 microliter/min. The blood flow rate per unit mass of retinal tissue was calculated and found to be in good agreement with that reported for macaque monkeys. Blood flow to the temporal side of the retina was approximately three times larger than to the nasal side. There was no significant difference between blood flow to the superior and the inferior retina.