F. Schütt

Patterns of increased in vivo fundus autofluorescence in the junctional zone of geographic atrophy of the retinal pigment epithelium associated with age-related macular degeneration

Abstract · Purpose: To determine in vivo lipofuscin (LF)-induced topographic variations of fundus autofluorescence in eyes with geographic atrophy (GA) of the retinal pigment epithelium (RPE) associated with age-related macular degeneration (ARMD). · Methods: Fundus autofluorescence was examined with a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph) after excitation with an argon laser (488 nm) and detection of the emitted light above 500 nm. Fifty-seven eyes of 38 patients with uni- or multifocal GA associated with ARMD were studied. The findings were compared with 43 eyes with GA secondary to other etiologies, including juvenile macular dystrophies. · Results: An increased autofluorescence outside the GA was observed in 47 (82.5%) of 57 eyes with GA associated with ARMD in contrast to 4 (9.3%) of 43 eyes with GA of other causes (P<0.001). Three different patterns were noted: a continuous band at the margin with variable peripheral extension in 36 eyes (76.6%), a diffusely increased autofluorescence at the entire posterior pole in 6 eyes (12.8%), and small focal spots of increased autofluorescence in the junctional zone in 3 eyes (6.4%). Of 19 patients with bilateral GA, 17 (89.5%) had an identical pattern in both eyes. · Conclusions: The different patterns of autofluorescence in the presence of GA associated with ARMD may reflect variable forms of reactive changes in the surrounding RPE cells, and may indicate the extend of compromised RPE secondary to ageing changes in the outer retina, Bruch’s membrane and choriocapillaris. Since GA spreads over time, increased LF accumulation in the junctional zone may precede cell death and may, therefore, be of prognostic value. Knowledge of the topographic variation in LF accumulation is important because heterogeneity may reflect underlying differences in cell kinetics, metabolism and biochemistry.

Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration

Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases, including age‐related macular degeneration (AMD). The major lipofuscin fluorophor A2‐E is thought to play an important pathogenetic role. In previous studies A2‐E was shown to severely impair lysosomal function of RPE cells. However, the underlying molecular mechanism remained obscure. Using purified lysosomes from RPE cells we now demonstrate that A2‐E is a potent inhibitor of the ATP‐driven proton pump located in the lysosomal membrane. Such inhibition of proton transport to the lysosomal lumen results in an increase of the lysosomal pH with subsequent inhibition of lysosomal hydrolases. An essential task of the lysosomal apparatus of postmitotic RPE for normal photoreceptor function is phagocytosis and degradation of membranous discs shed from photoreceptor outer segments (POS) and of biomolecules from autophagy. When the lysosomes of cultured RPE cells were experimentally loaded with A2‐E, we observed intracellular accumulation of exogenously added POS with subsequent congestion of the phagocytic process. Moreover, the autophagic sequestration of cytoplasmic material was also markedly reduced after A2‐E loading. These data support the hypothesis that A2‐E‐induced lysosomal dysfunction contributes to the pathogenesis of AMD and other retinal diseases associated with excessive lipofuscin accumulation.

Proteome analysis of lipofuscin in human retinal pigment epithelial cells

Excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial cells is a common pathogenetic pathway in various blinding retinal diseases including age‐related macular degeneration, which is now the most common cause of registerable blindness in the industrialized nations. To better understand the role of lipofuscin accumulation and to manipulate the pathogenetic mechanisms on both experimental and therapeutic levels we analyzed the proteome of isolated human ocular lipofuscin granules from human RPE cells. After homogenization and fractionation by gradient ultracentrifugation of the RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix‐assisted laser desorption/ionization mass spectrometry and HPLC‐coupled electrospray tandem mass spectrometry. Besides a better understanding of downstream pathways, this approach may provide new targets for therapeutic interventions in a currently untreatable disease.

Agreement among ophthalmologists in evaluating fluorescein angiograms in patients with neovascular age-related macular degeneration for photodynamic therapy eligibility (FLAP-study)

OBJECTIVE To determine intraobserver and interobserver variation for classifying types of choroidal neovascularizations (CNV) in exudative age-related macular degeneration (ARMD). DESIGN Intraexaminer and interexaminer reliability study. PARTICIPANTS Digital high-quality fluorescein angiograms of 40 patients with neovascular ARMD were evaluated independently by 16 retinal specialists. MAIN OUTCOME MEASURES Fluorescein angiographies were presented in two randomized sequences (series A and B) to each masked reader for classification of type of CNV into classic, occult, or mixed with classic component of less or greater 50%. Agreement was evaluated by calculating kappa statistics (kappa) and intraclass correlation coefficients. RESULTS The mean kappa coefficient was 0.64 +/- 0.11 for intraobserver variation, with a range from 0.44 to 0.89. For interobserver variation the intraclass correlation coefficients was calculated as 0.66 (95% confidence interval [CI] 0.56, 0.77) for series A and as 0.55 (95% CI 0.43, 0.67) for series B. CONCLUSIONS Angiographic classification of CNV secondary to ARMD can vary considerably not only between observers but also for repeated evaluation by the same observer. Because various current and emerging treatments including photodynamic therapy are based on specific angiographic characteristics, accurate interpretation will become more important.

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

PURPOSE To describe the development of new and enlargement of preexisting atrophy confined to areas with abnormally high levels of in vivo autofluorescence in eyes with geographic atrophy (GA) associated with age-related macular degeneration (ARMD). METHODS The spatial distribution and intensity of fundus autofluorescence as well as the spread of GA and occurrence of new GA was recorded over a period of 3 years in three patients with ARMD using a confocal scanning laser ophthalmoscope. RESULTS A diffuse irregular increased autofluorescence at the posterior pole was recorded at baseline in the presence of unifocal or multifocal patches of geographic atrophy. Within these areas of elevated autofluorescence, new atrophic areas developed, and existing patches of atrophy enlarged during the review period, whereas this was not observed in areas with normal background autofluorescence. The total area of abnormal autofluorescence also showed enlargement over time. CONCLUSIONS These preliminary findings suggest that areas of increased autofluorescence precede the development and enlargement of outer retinal atrophy in eyes with ARMD. Because the dominant fluorophores of fundus autofluorescence are part of lipofuscin granules of RPE cells, the observations indicate that excessive RPE lipofuscin accumulation may be of significance in the pathogenesis of GA associated with ARMD. With GA being a major cause for severe visual loss in ARMD, in vivo fundus autofluorescence recording over time may allow identification of prognostic determinants and may give important clues to the understanding of mechanisms of disease.

Electrically induced vasomotor responses and their propagation in rat renal vessels In vivo

1 Vasomotor responses (VMR) induced by local electrical stimulation were studied in the vasculature of the split hydronephrotic rat kidney by in vivo microscopy. 2 Unipolar pulses, which were applied by a micropipette positioned close to the vessel wall, elicited local and propagated VMR. Depolarizing and hyperpolarizing currents caused vaso‐constriction and vasodilatation, respectively. 3 The magnitude of VMR could be controlled within seconds by variation of pulse frequency, pulse width and voltage. VMR were abolished by slight retraction of the stimulating micro‐pipette. Repetitive electrical stimulation resulted in reproducibly uniform VMR. 4 Propagated VMR decayed with increasing distance from the stimulation site. They decayed more rapidly in the upstream than in the downstream flow direction in interlobular arteries. The longitudinal decay was well approximated by an exponential function with significantly different length constants of 150 ± 40 μm (upstream, n= 5) and 420 ± 90 μm (downstream, n=8). 5 Our results show that vasomotor responses, which are initiated by changes in membrane potential, are propagated over distances of potential physiological importance in interlobular arteries.

Moderately Reduced ATP Levels Promote Oxidative Stress and Debilitate Autophagic and Phagocytic Capacities in Human RPE Cells

PURPOSE Aging of the RPE is associated with a decrease of intracellular ATP levels and increased oxidative stress. We investigated the effects of moderate energy deficit on intracellular glutathione levels, oxidative damage of cellular proteins and DNA, and autophagy rates using an RPE cell culture model. Additionally, phagocytosis of photoreceptor outer segments was assayed as an example of an ATP-dependent normal function of the RPE. METHODS ATP synthesis of primary human RPE cells was moderately inhibited by atractyloside. Oxidative stress was induced by tert-butyl hydroperoxide (tBH). ATP, reduced glutathione (rG), malondialdehyde (MDA) adduct formation and 8-hydroxydeoxyguanosine (8OHdG) levels were measured. Autophagy and phagocytosis of photoreceptor outer segments were assayed by radiometric methods. RESULTS Atractyloside-treatment reduced cellular ATP levels by 30%, mimicking the energy status of aged RPE. tBH decreased rG in RPE cells with lowered ATP levels whereas cells with normal ATP content were not affected. tBH-induced oxidative stress resulted in substantial accumulation of MDA protein adducts in cells with lowered ATP while cells with regular ATP levels were only modestly affected. tBH induced more oxidative DNA damage (8OHdG formation) in cells with lowered ATP levels than in cells with regular ATP. In atractyloside-treated cells, autophagy rates decreased 3-fold as compared with controls. Phagocytic capacity for uptake and degradation of photoreceptor segments was reduced in RPE with low ATP. CONCLUSIONS Moderately decreased ATP levels such as seen in aged individuals might contribute to the vulnerability of RPE to oxidative stress damage and to dysfunction.

Temperature dependent fluorescence of A2-E, the main fluorescent lipofuscin component in the RPE

Purpose. A2-E is the dominant fluorophore of lipofuscin in the retinal pigment epithelium. In an in-vitro setup, we determined the temperature-dependent changes of the A2-E fluorescence with the aim of also assessing the potential value of such measurements for determining retinal temperature by autofluorescence measurements during laser treatment. Methods. A2-E was biosynthesized and diluted in Dimethyl Sulfoxide (DMSO) to 1 µM. Fluorescence measurements were performed with a photospectrometer under various temperatures ranging from 20°C to 75°C. Autofluorescence was excited at 467 nm, and emission was detected around 632 nm. Results. A2-E fluorescence intensity showed a linear decrease concomitant with temperature increment. At 75°C, the fluorescence intensity decreased by 43% compared to at 20°C. Fluorescence intensity was completely reversible dependent on the temperature, which cannot be explained by thermal A2-E alteration. Conclusions. If the A2-E temperature-dependent fluorescence in-vitro is transferable to human fundus auto-fluorescence, then it may be possible to apply an autofluorescence-based online detection device for noninvasive determination of fundus temperature during in vivo laser treatment. This is of clinical relevance, especially for the application of photodynamic therapy (PDT) and transpupillary thermotherpy (TTT).

Indocyanine green angiography in the presence of subretinal or intraretinal haemorrhages: clinical and experimental investigations

Purpose: The absorption and emission characteristics of indocyanine green are associated with better penetration through ocular pigments, including melanin and blood, in comparison with fluorescein. Therefore, it has been assumed that indocyanine green angiography (ICG‐A) allows better delineation of fluorescent structures including choroidal neovascularization in the presence of haemorrhages. The degree and frequency of blockage by haemorrhages during ICG‐A and fluorescein angiography (Fl‐A) were compared and absorption characteristics by blood were experimentally determined.

[OCT-guided reinjection of 2.5 mg bevacizumab for treating macular edema due to retinal vein occlusion].

BACKGROUND Macular edema (ME) due to retinal vein occlusion can be successfully treated with intravitreal bevacizumab therapy. There is no common recommendation concerning time intervals and criteria for reinjection. METHOD Sixty-three patients (follow-up 30+/-18 weeks) received intravitreal injections of 2.5 mg bevacizumab. Reinjection was performed only if optical coherence tomography (OCT) showed persistent or recurrent ME. Check-ups were performed every 6-8 weeks. RESULTS There was complete resolution of macular edema in 31 patients after the first injection (improvement in visual acuity 3.7+/-3.7 lines); 65.2% of these patients developed recurrence of ME within 13.3+/-4.4 weeks, which completely resolved again after a second injection. Visual acuity gained the same level as after the first injection. Another relapse of ME in this group occurred in 69% of patients after another 13.4+/-5.4 weeks. Patients with persistent ME after the first injection (n=32) received a second injection, initially leading to resolution of ME in 33.3%, but all of these patients had a relapse within 13.9+/-4.1 weeks. CONCLUSION OCT-guided reinjection leads to anatomic and functional stabilization or improvement even if transient recurrence of ME occurs.