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The universal energy source adenosine triphosphate (ATP)is reduced by approximately 30 % in the retinal pigment epithelium (RPE) of elderly persons. Increased oxidative stress and decreased antioxidative capacity, such as glutathione in aging eyes cause impairment of energy-dependent RPE processes and lead to loss of visual function. We developed a cell culture model of aging RPE using atractyloside to inhibit mitochondrial ATP synthesis and tert-butyl hydroperoxide as oxidant. The ATP levels were reduced by 30 % and oxidative damaged proteins and DNA increased whereas antioxidative glutathione decreased. Autophagy as an internal cellular repair mechanism and phagocytosis of photoreceptors were impaired. Antioxidative and mitochondria-activating Ginkgo biloba extract EGb 761 increased the intracellular ATP level and antioxidative glutathione. This cell culture model seems to be suitable to investigate in vitro the effect of protective substances and their compounds on aging processes in RPE.

Ocular fundus photography allows detection of both ocular and systemic diseases. This study investigated the efficacy of a broad screening in a department of internal medicine using nonmydriatic digital fundus photography. For 8 weeks a medical technician was trained in using the camera as well as interpreting the photographs. The medical technician and an ophthalmologist evaluated the fundus photographs separately by using a self-developed questionnaire. The fundus camera was user-friendly and after several weeks of adjustment and practical application the medical technician was able to detect the majority of pathological fundus photographs. Out of 218 patients examined 148 (68%) were identified as pathological by the medical technician and 163 (75%) by the ophthalmologist (p = 0.0003). The medical technician missed 15 (7%) patients. Furthermore the diagnoses made by the medical technician were faulty. In summary an ophthalmological screening by a medical technician is feasible but the diagnosis still remains the responsibility of ophthalmologists. Such a compromise could facilitate the examination of a large number of patients and disclose previously unrecognized diseases.

Purpose: The aim of this study was to evaluate the safety and efficacy of intravitreal bevacizumab as a treatment for choroidal neovascularization (mCNV) due to pathological myopia., Methods: A consecutive series of 23 patients with mCNV treated with 1.25 mg intravitreal bevacizumab after being thoroughly informed about the off-label character of the treatment. Of the patients 6 received additional photodynamic therapy at the time of first injection. Reinjections were given every 6 weeks if intraretinal or subretinal fluid persisted., Results: The pre-injection mean visual acuity (VA) was 0.25 (0.58 logMAR+/-0.36). During a follow up of 16.6+/-13.7 weeks 1.4+/-0.8 injections were given. Complete resorption of subretinal or intraretinal fluid was achieved in all patients. VA improved by 2.3+/-3.5 lines on average, 9 patients (39.1%) had an increase of >or=3 lines, none lost more than 1 line. Patients with bevacizumab monotherapy (n=17) had an improvement of 2.59+/-3.9 lines, 7 patients (41.2%) had an increase of >or=3 lines. No intraocular or systemic side effects were observed., Conclusions: In this as yet largest series of patients with mCNV treated with intravitreal bevacizumab, the treatment seemed to be effective and safe.

Background: We present an optical coherence tomography (OCT)-based individual reinjection procedure for bevacizumab treatment in neovascular age-related macular degeneration (ARMD)., Methods: Thirty-two patients with active subfoveal occult choroidal neovascularisation in ARMD received a single intravitreal injection of 1.25 mg bevacizumab and were reinjected based on new or persisting subretinal or intraretinal fluid on OCT. Patient visits were every 6-8 weeks., Results: After a single injection, 74% of patients demonstrated complete retinal fluid absorption, with 44% of patients showing no relapse during a follow-up of 30+/-13 weeks. Fifty-six percent of patients required a second injection after a mean of 19+/-8 weeks, with 82% of patients showing absorption of macular fluid thereafter with regain of their previous achieved best-corrected visual acuity. Thirty-two percent did not require any further injection (follow-up 32+/-12 weeks). Of those patients not showing retinal fluid absorption after the first injection (26%), 44% demonstrated retinal fluid absorption after the second injection. All patients achieved stabilisation of visual acuity during follow-up, with 30% of patients showing a significant gain of >or=3 lines., Conclusions: OCT-based reinjections of bevacizumab in neovascular ARMD reduce the number of injections and lead to anatomic and functional retinal stabilisation.

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.

Purpose: To report on the efficacy of intravitreal bevacizumab as off-label therapy in different angiographic subtypes in neovascular age-related macular degeneration (AMD)., Methods: Seventy-five patients with neovascular AMD and recent disease progression were classified into different angiographic subtypes and were treated with intravitreal bevacizumab (1.25 mg/0.05 ml) at 6-week intervals. Patients with subfoveal classic choroidal neovascularization (CNV) also received photodynamic therapy. ETDRS visual acuity, ophthalmic exams, and optic coherence tomography (OCT) were performed before treatment, 1 week after treatment, and then on a 6-week basis. Fluorescein angiographies and medical check-ups were also done., Results: Bevacizumab led to stabilization of visual acuity (loss of less than 15 letters) in all angiographic subtypes during a follow-up of 37+/-13 weeks. Patients with occult extrafoveal CNV (n=6) profited the most and gained 2+/-2 lines. Treatment with intravitreal bevacizumab was very well tolerated in all patients, with neither systemic nor intraocular side effects, with the exception of one retinal pigment epithelium tear., Conclusion: Intravitreal bevacizumab treatment is efficacious in all angiographic CNV subtypes and leads to reduction of macular edema and stabilization or improvement in visual acuity.

Background: Retinal vein occlusion often leads to macular edema as a result of an elevated level of intravitreal VEGF. We report on the anatomic and functional results after intravitreal bevacizumab injections in patients with retinal vein occlusion., Methods: In a prospective study, 18 patients with central, and 22 patients with branch retinal vein occlusion, all of whom had persistent macular edema (>300 microm) received 2.5 mg intravitreal bevacizumab. ETDRS visual acuity, ophthalmic examination and stratus OCT were performed at baseline, 1 week after injection and then monthly. Further injections were given every 6 weeks in patients with persistent or recurring macular edema., Results: The findings did not deteriorate in any of the 40 patients. The injections (mean of 2.6+/-1.4 injections/patient) were very well tolerated in all cases during a mean follow-up of 23+/-13 weeks. On the last visit, 73.3% of patients with central retinal vein occlusion and 76.5% of those with branch retinal vein occlusion were found to have significantly improved visual acuity (by at least 3 lines). Mean central retinal thickness had decreased from 921+/-264 to 239+/-66.2 microm in patients with central retinal vein occlusion, and from 678+/-221 to 236+/-78 microm in patients with branch retinal vein occlusion., Conclusions: Neither intraocular nor systemic side-effects were observed in this study after repeated intravitreal injections of 2.5 mg bevacizumab. Current results suggest that intravitreal anti-VEGF therapy is a promising option in macular edema secondary to retinal vein occlusion.

Background: Accumulation of lipofuscin in RPE cells occurs with age and in association with various retinal diseases. Lipofuscin and its major retinoid compound and fluorophore A2-E interfere with the cellular metabolism of RPE cells in various ways. One of these mechanisms is thought to be related to detergent properties of A2-E., Methods: We isolated pure and intact lysosomes from RPE cell cultures and investigated detergent-like effects of the lipofuscin compound A2-E on the integrity of lysosomal membrane and other cellular membranes, using latency measurements. A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined., Results: A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved upon incubations in isotonic media providing the base for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison, leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. The described methodology to obtain highly purified and intact lysosomes from RPE cells provides a suitable tool for investigations on compounds affecting lysosomal structure. A2-E was shown to cause desintegration of the lysosomal membrane at relatively low concentrations, which may implicate an involvement of such mechanism in triggering lipofuscin-induced dysfunction of RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism., Conclusions: Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin-accumulation, such as age-related macular degeneration.

Purpose: Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements., Methods: A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker beta-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined., Results: A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 microM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 microM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid., Conclusions: The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.

Background: Excessive accumulation of lipofuscin in the retinal pigment epithelium with age and in various hereditary and degenerative retinal diseases, is of pathogenetic significance. We have shown that the major lipofuscin fluorophor A2-E (N-retinylidene-N-retinylethanolamine) affects the lysosomal degradation of human RPE cells and damages the cellular metabolism by phototoxic properties. Herein we sought to determine mechanisms for the inhibitory effect on lysosomal function apart from pH elevation., Methods: Potter-Elvejem homogenates of RPE cells were used to measure the activity of 24 lysosomal enzymes before and after incubation with A2-E., Results: This is the first time that RPE cells have been screened for a large spectrum of lysosomal hydrolases including proteases, lipidases, glycosidases, nucleases, sulfatases and phosphatases. The activities of these hydrolases were readily detectable in cultured RPE cells. Incubation of RPE cell homogenates even with high A2-E concentrations (up to 10 microM) did not affect the activity of isolated lysosomal enzymes., Conclusions: The results suggest that a direct inhibition of lysosomal enzyme activity would not explain the inhibitory effect on lysosomal degradation. A2-E increases the acidic intralysosomal pH thereby probably hindering pH-dependent lysosomal enzymatic activities. The understanding of the inhibitory effects of A2-E on RPE cell metabolism may contribute to new approaches for treatment of retinal diseases with excessive lipofuscin accumulation such as ARMD or M. Stargardt.

Background: A fluorescent compound of lipofuscin, A2-E, has been shown to impair lysosomal function and to increase the intralysosomal pH of human retinal pigment epithelial (RPE) cells. This study addressed the phototoxic potential of A2-E on RPE cells., Methods: A2-E accumulation was confirmed by fluorescence microscopy and fluorescence-activated cell sorter analysis. Acridine orange staining allowed assessment of lysosomal integrity and intralysosomal pH. Phototoxic properties of A2-E were determined by exposing A2-E-free and A2-E-fed RPE cell cultures to short-wavelength visible light and assessing cell viability and lysosomal integrity., Results: Intralysosomal accumulation of A2-E was confirmed. Acridine orange staining showed that the A2-E was located in the lysosomal compartment and induced an elevation of intralysosomal pH. Exposure of A2-E fed cells to light resulted in a significant loss of cell viability by 72 h which was not observed in either RPE cells maintained in the dark or A2-E-free cultures exposed to light. Toxicity was associated with a loss of lysosomal integrity., Conclusion: A2-E is detrimental to RPE cell function by a variety of mechanisms including inhibition of lysosomal degrading capacity, loss of membrane integrity, and phototoxicity. Such mechanisms could contribute to retinal aging and to retinal diseases associated with excessive lipofuscin accumulation.

Purpose: Lipofuscin accumulates with age and in association with various retinal diseases. To investigate cellular effects of lipofuscin components in an in vitro RPE cell model, specific loading of the lysosomal compartment is required. Herein a major lipofuscin fluorophor was complexed to LDL and the subsequent subcellular localization of the retinoid was examined., Methods: The lipofuscin component N-retinylidene-N-retinylethanolamine (A2-E) was synthesized and coupled to LDL. Human RPE cell cultures were loaded with the A2-E/LDL complex over 4 weeks. Thereafter, RPE cells were harvested by trypsinization and disrupted by nitrogen cavitation. After ultra-centrifugation, the postnuclear supernatant was fractionated on a self-generating gradient and fractions were analyzed by measuring marker enzyme activities of various cellular compartments., Results: A2-Eaccumulated almost exclusively in the lysosomal compartment, as indicated by the identical peaks of the marker enzyme ss-hexosaminidase and the relative fluorescence of A2-E. Only a small amount of A2-E appeared to associate with the cell membrane, as shown by a minor peak of A2-E corresponding to the distribution of phosphodiesterase activity. The lysosomal marker enzyme was not present in the cytosolic fraction., Conclusions: The feeding of A2-E/LDL complexes to cultured RPE-cells proved to be highly effective in specific loading of the lysosomal compartment, providing a suitable in vitro cell culture model for RPE aging and the investigation of A2-E-effects on lysosomal functions in RPE cells. Such a model may contribute to the understanding of the pathogenesis of degenerative diseases of the outer retina associated with excessive lipofuscin accumulation, including age-related macular degeneration, Stargardt's disease and Best's disease.

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.