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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.

Hintergrund Untersuchung von Sicherheit und Effektivitat von intravitrealem Bevacizumab bei choroidaler Neovaskularisation infolge pathologischer Myopie (mCNV).

Untersucht wird ein OCT-abhangiges individuelles Reinjektionsprozedere mit intravitrealem Bevacizumab bei neovaskularer AMD. 32 Patienten mit aktiver subfovealer okkulter CNV bei AMD wurden einmalig mit 1,25 mg Bevacizumab intravitreal behandelt und nur reinjiziert bei im OCT persistierender bzw. erneut auftretender sub- bzw. intraretinaler Flussigkeit. Kontrolluntersuchungen erfolgten in 6–8-wochigen Abstanden. Nach einmaliger Injektion war bei 74% der Patienten die sub- und intraretinale Flussigkeit komplett resorbiert. 44% dieser Patienten blieben wahrend eines Follow-up von 30±13 Wochen trocken, 56% benotigten nach durchschnittlich 19±8 Wochen eine 2. Injektion, wonach bei 82% der Befund erneut abtrocknete und sich der initiale Visusgewinn wieder einstellte. 32% dieser Patienten blieben daraufhin stabil trocken (Follow-up 32±12 Wochen). Von den Patienten, die nach der 1. Injektion nicht abtrockneten (26%), zeigte sich bei 44% nach der 2. Injektion ein trockener Befund. Bei allen Patienten konnte uber die Behandlungsdauer eine Visusstabilisierung erzielt werden, bei 30% ein signifikanter Visusanstieg ≥3 Linien. Bevacizumab-Reinjektionen in Abhangigkeit vom OCT-Befund fuhren bei exudativer AMD neben einer Reduktion der Injektionsanzahl zu einer anatomischen und funktionellen Befundstabilisierung.

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.

Hintergrund Das Makulaodem (MO) infolge venoser retinaler Verschlusse lasst sich durch intravitreales Bevacizumab gut beeinflussen. Unklar ist, wann und nach welchen Kriterien Reinjektionen erfolgen sollten.

Untersucht wurde der Effekt von intravitreal appliziertem Bevacizumab als Off-Label-Therapie bei verschiedenen angiographischen Subtypen der neovaskularen altersabhangigen Makuladegeneration (AMD). 75 Patienten mit neovaskularer AMD und „recent disease progression“ wurden angiographischen Subtypen zugeordnet und mit intravitrealem Bevacizumab (1,25 mg/0,05 ml) in 6-wochigen Abstanden therapiert. Bei subfovealer klassischer choroidaler Neovaskularisation (CNV) erfolgte zusatzlich eine photodynamische Therapie. ETDRS-Visus, ophthalmologische Untersuchungen und optische Koharenztomographien (OCT) wurden vor Therapie, nach einer Woche und in 6-wochigen Intervallen durchgefuhrt. Weiterhin erfolgen Fluoreszenzangiographien und internistische Kontrolluntersuchungen. Bei allen angiographischen Subtypen fuhrte die Bevacizumab-Therapie wahrend einer Nachbeobachtungszeit von 37±13 Wochen im Mittel zu einer Visusstabilisierung (Verlust von weniger als 15 Buchstaben). Patienten mit okkulter extrafovealer CNV (n=6) erzielten den hochsten Visusanstieg (2±2 Linien). Die Bevacizumab-Therapie wurde von allen Patienten ohne systemische oder intraokulare Nebenwirkungen sehr gut vertragen. In einem Fall kam es zum Riss des retinalen Pigmentepithels. Intravitreale Bevacizumab-Injektionen wirken bei allen angiographischen CNV-Subtypen im Sinne einer Makulaodemreduktion und fuhren zu einer Visusstabilisierung bzw. -besserung.

BACKGROUND: Gorlin Goltz syndrome is a rare, autosomal dominant inherited disease that is characterised by multiple basal cell carcinomas (BCC) including the periorbital region and eye lids. We report a severe infantile manifestation with lid involvement treated by photodynamic therapy (PDT). PATIENT: A 13-year-old boy with Gorlin Goltz syndrome presented with multiple confluent BCC on both eye lids and the skin of neck and trunk. Multiple bilateral periorbital confluent and surgically not removable BCC were treated by topical PDT. RESULTS: Numerous superficial BCC were successfully treated by photodynamic therapy with remarkable cosmetic results. CONCLUSION: In cases of numerous confluent and surgically not removable BCC, PDT represents an effective therapy. Frequent monitoring is necessary to maintain the clinical outcome.

Background In the retinal pigment epithelium (RPE) lipofuscin granules accumulate with age in the lysosomal compartment mainly as a byproduct of constant phagocytosis of oxidized membranous discs shed from photoreceptor outer segments. Antioxidative defiency and prooxidative conditions in the RPE play a key role in the pathogenesis of RPE dysfunction and macular degenerations such as ARMD. In human RPE cell cultures we investigated the antioxidative effect of N-acetylcysteine (ACC) on lysosomal functions. Methods Primary human RPE cell cultures were loaded with regular or oxidized human and porcine rod outer segments (ROS) and treated with ACC. Lysosomal volume and accumulation of autofluorescent material was measured using [14C] methylamine accumulation and FACS analysis. The regulation pattern of lysosomal proteins were investigated by proteome analysis. Results ACC reduced total lysosomal volume in control, ROS and oxidized ROS fed RPE cells. After ROS incubation increased accumulation of autofluorescent material was measured. ACC treatment decreased intracellular accumulation. Furthermore, incubation with ACC leads to a general down regulation of lysosomal proteins. Conclusion In our cell culture model of ROS fed RPE cells simulating aged RPE ACC improves lysosomal volume and metabolism. Therefore ACC may represent a new prophylactic and causal treatment option for AMD.

Venose retinale Gefasverschlusse konnen durch eine erhohte intravitreale VEGF-Konzentration zur Ausbildung eines Makulaodems fuhren. Wir berichten uber anatomische und funktionelle Ergebnisse nach intravitrealer Bevacizumab-Injektion bei venosen retinalen Verschlussen. 18 Patienten mit Zentralvenen- und 22 Patienten mit Venenastverschluss und persistierendem Makulaodem (>300 μm) wurden in einer prospektiven Studie mit 2,5 mg Bevacizumab intravitreal behandelt. EDTRS-Visus, ophthalmologische Untersuchung und OCT erfolgen vor Therapiebeginn, 1 Woche nach Injektion und in monatlichen Intervallen. Reinjektionen wurden bei persistierendem bzw. erneutem Makulaodem in 6-wochigen Abstanden durchgefuhrt. Bei keinem der 40 Patienten kam es zu einer subjektiven oder objektiven Befundverschlechterung. Die Injektionen (im Mittel 2,6±1,4 Injektionen) wurden bei einem mittleren Follow-up von 23±13 Wochen ausnahmslos sehr gut vertragen. Bei 73,3% der Patienten mit Zentralvenen- und bei 76,5% der Patienten mit Venenastverschluss kam es bei letzter Vorstellung zu einem signifikanten Visusanstieg (Visusgewinn um mindestes 3 Zeilen). Gleichzeitig hat sich die mittlere Netzhautdicke bei Patienten mit Zentralvenenverschluss von 921±264 μm auf 239±66,2 μm, und bei Patienten mit Venenastverschluss von 678±221 auf zuletzt 236±78 μm reduziert. In dieser Studie konnten nach intravitrealen Bevacizumab-Injektionen in einer Konzentration von 2,5 mg bislang keine intraokularen oder systemischen Nebenwirkungen beobachtet werden. Die bisherigen Ergebnisse zeigen, dass die intravitreale Anti-VEGF-Therapie eine viel versprechende Option bei Makulaodem infolge venoser retinaler Verschlusse ist.

Lipofuscin occurs in association with various blinding diseases, including ARMD. Formation of lipofuscin is considered to be initiated by the inability of the RPE lysosome to degrade constituents of phagocytosed material resulting in its intralysosomal accumulation. Thus, the deposition of abnormal retinoid adducts causing the autofluorescent properties of RPE lipofuscin originates from abnormal products of the retinoid cycle contained in phagocytosed photoreceptor outer segments. The major lipofuscin retinoid conjugate A2-E was previously shown to exert toxic effects on RPE cells by directly damaging lysosomal function and structure. However, A2-E was also proposed to severely harm extralysosomal RPE cell structures during the pathogenesis of ARMD. This would require release or leakage of A2-E from the lysosomal compartment with subsequent targeting of other cellular compartments.We therefore now investigated intralysosomal accumulation, possible biodegradation, release from the lysosomal compartment and intracellular spreading of (14)C-labelled A2-E in cultured human RPE cells. We specifically loaded lysosomes of cultured human RPE cells with [(14)C]A2-E.A linear increase of intracellular radioactivity was observed during the 4-week loading period. Cell fractionation experiments indicated that more than 90% of loaded A2-E was specifically accumulating in the lysosomes. After loading, the fate of the radioactive label was chased over a period of an additional 4 weeks. No metabolism or secretion of A2-E to the medium was detectable. Subcellular fractionation revealed that during the chase period, about 13% were shifted from the lysosomes to mitochondrial fractions. This effect was strikingly intensified when after loading the cells with the labeled retinoid, its intralysosomal concentration was boosted by an additional load with non-labeled A2-E. Thus about 44% of the label were located in mitochondria at the end of the chase period. No significant spreading to other cell compartments was detectable.Since A2-E was suggested to act as a proapoptotic molecule via a mitochondrial pathway, we postulate that upon reaching a critical intralysosomal concentration, A2-E is released from the lysosome and then specifically targets the outer mitochondrial membrane thereby initiating apoptosis of the RPE cell. This may also apply correspondingly to other lipofuscin-associated molecules that cause leakage of the lysosomal membrane.

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.A2-E was biosynthesized and diluted in Dimethyl Sulfoxide (DMSO) to 1 microM. Fluorescence measurements were performed with a photospectrometer under various temperatures ranging from 20 degrees C to 75 degrees C. Autofluorescence was excited at 467 nm, and emission was detected around 632 nm.A2-E fluorescence intensity showed a linear decrease concomitant with temperature increment. At 75 degrees C, the fluorescence intensity decreased by 43% compared to at 20 degrees C. Fluorescence intensity was completely reversible dependent on the temperature, which cannot be explained by thermal A2-E alteration.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).

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 (κ) and intraclass correlation coefficients. Results The mean κ 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.

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 β-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 µM A2-E, progressive leakage of the lysosomal marker was observed. In comparison, leakage of the mitochondrial marker was induced at significantly lower concentrations (1µM), 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.

Hintergrund. Lipofuszin akkumuliert in postmitotischen retinalen Pigmentepithelzellen (RPE) mit dem Alter und in Assoziation mit verschiedenen Makula-/Netzhautdegenerationen. Wir konnten zeigen, dass der Lipofuszinretinoid-Bestandteil A2-E (N-Retinyliden-N-Retinyl-Ethanolamin) lysosomale Funktionen hemmt. Um weitere Wirkungsmechanismen fur toxische Effekte von A2-E zu evaluieren, untersuchten wir nach Etablierung einer Methode zur Isolation intakter Lysosomen aus RPE-Zellkulturen Detergenzieneffekte auf lysosomale und andere zellulare Membranen mittels Latenzmessungen. Methoden. Zur Isolation intakter Lysosomen wurde der postnukleare Uberstand humaner RPE-Zellen mittels sequenzieller Dichtegradientenzentrifugation fraktioniert. Die Ruptur der lysosomalen Membran wurde durch Latenzmessungen des lysosomalen Leitenzyms β-Hexosaminidase an mit A2-E inkubierten isolierten Lysosomen erfasst. Latenzen der Markerenzyme Succinatdehydrogenase and UDP-Galactosyltransferase zeigen Effekte des A2-E auf Membranen isolierter Mitochondrien und Mikrosomen. Nach Zugabe von A2-E in das Kulturmedium konnten durch Messung der Laktatdehydrogenase Schaden der Plasmamembran erfasst werden. Ergebnisse. Bis auf eine geringe Restaktivitat mitochondrialer Leitenzyme konnte eine 100fache Anreicherung der lysosomalen Fraktion erzielt werden. Eine Kontamination mit anderen Zellfraktionen wurde nicht beobachtet. In geeigneten isotonischen Puffern besitzen die Lysosomen eine ausreichende Stabilitat, um mogliche Detergenzieneffekte des A2-E auf lysosomale Membranen nachzuweisen. Bei Konzentrationen uber 2 μM A2-E kam es zur Leckage des lysosomalen Leitenzyms. Leckage des mitochondrialen Leitenzyms erfolgte bereits ab 1 μM A2-E, wahrend ER/Golgi-Membranen und Plasmamembranen relativ resistent gegenuber Detergenzieneffekten von A2-E waren. Schlussfolgerung. Die beschriebene Methode zur Isolation intakter Lysosomen aus RPE-Zellen ist auch fur zukunftige Untersuchungen fur lysosomale Effekte verschiedener Biomolekule nutzbar. A2-E induziert bereits in niedriger Konzentration eine Desintegration der lysosomalen Membran. Detergenzieneffekte an der mitochondrialen Membran stellen offensichtlich einen weiteren Pathomechanismus dar, wodurch Reaktionskaskaden, die letztlich zum Zelltod fuhren konnen, induziert werden. Unsere Beobachtungen sprechen fur einen Detergenzieneffekt von A2-E an verschiedenen zellularen Membranen, der bei allen retinalen Erkrankungen mit exzessiver Lipofuszinakkumulation, wie der AMD, von Bedeutung sein konnte. Ein besseres Verstandnis dieser molekularen Mechanismen eroffnet neue therapeutische Perspektiven.

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. Methods: Simultaneous confocal scanning laser ophthalmo­scopy was performed in patients with intraretinal or sub­retinal haemorrhages associated with various retinal diseases including neovascular age-related macular degeneration. Areas of blocked choroidal fluorescence were compared in Fl-A and ICG-A using a standardized classification system by two independent readers. Experimental absorption measurements were performed using blood-filled quartz cuvettes and laser light with 488 and 790 nm, respectively. Results: Sixty eyes of 59 patients were analysed. Twelve eyes (20%) showed blockage in Fl-A only corresponding with funduscopically visible blood. In 35 eyes (58%) the extent of absorption was greater in Fl-A compared with ICG-A. An identical area of blockage in both Fl-A and ICG-A was noted in 13 eyes (22%). The coefficient of absorption was 18.4 mm-1 for Fl-A (488 nm) and 5.4 mm-1 for ICG-A (790 nm). Conclusions: In contrast to previous assumptions, the findings indicate that clinically intraretinal or subretinal haemorrhages are frequently associated with blockage not only in Fl-A but also in ICG-A. This is in accordance with the experimentally determined coefficient of absorption. Apparently, haemorrhages occurring in association with retinal and choroidal diseases commonly have a thickness sufficient enough to induce relevant absorption during ICG-A, and thus impair delineation of fluorescent structures in planes posterior to the haemorrhage. Therefore, the diagnostic value of ICG-A in presence of subretinal or intra­retinal bleedings is limited.

Hintergrund. Die exzessive Akkumulation von Lipofuszin in retinalen Pigmentepithelzellen ist sowohl bei der altersabhangigen Makuladegeneration (AMD) als auch bei verschiedenen erblichen Makuladystrophien von pathogenetischer Bedeutung. Wir haben gezeigt, dass der Lipofuszinfluorophor A2-E (N-Retinyliden-N-Retinyl-Ethanolamin) speziell den lysosomalen Stoffwechsel humaner retinaler RPE-Zellen hemmt und phototoxische Eigenschaften besitzt. Hier wurden neben der vorher gezeigten intralysosomalen pH-Wert-Erhohung weitere Mechanismen der Hemmung lysosomaler Enzyme untersucht. Methoden. RPE-Zellen wurden durch Potter-Elvejem-Homogenisation aufgeschlossen und in den resultierenden RPE-Zellhomogenaten mithilfe spezieller Enzymtests die Aktivitaten 24 lysosomaler Enzyme vor und nach Inkubation mit A2-E gemessen. Ergebnisse. In humanen RPE-Zellen wurde erstmals ein breites Spektrum lysosomaler Hydrolasen, das Proteasen, Lipasen, Glykosidasen, Nukleasen, Sulfatasen und Phosphatasen umfasst, gemessen. Lysosomale Enzymaktivitaten konnen hierbei in RPE-Zellen exakt und reproduzierbar bestimmt werden. Selbst hohe Konzentrationen von A2-E (10 μM) zeigten keine hemmende Wirkung auf lysosomale Hydrolasen im RPE. Schlussfolgerung. Mit den hier verwendeten Methoden lassen sich reproduzierbar lysosomale Enzymaktivitaten in humanen RPE-Zellen bestimmen. Die A2-E bedingte Hemmung des lysosomalen Stoffwechsels wird offensichtlich nicht durch eine direkte Enzymhemmung vermittelt. Ursachlich scheint ein intralysosomaler pH-Anstieg mit konsekutivem Verlust der lysosomalen Enzymaktivitat zu sein. Fur diese pH-Verschiebung konnte eine inhibitorische Wirkung auf die membranstandige Protonenpumpe verantwortlich sein. Das genaue Verstandnis der Hemmungsmechanismen von A2-E auf die Funktion von RPE-Zellen kann neue Wege der gezielten pharmakologischen Intervention bei Erkrankungen mit exzessiver Lipofuszinakkumulation aufzeigen.

Hintergrund. A2-E, ein fluoreszierender Bestandteil von Lipofuszin, beeintrachtigt den lysosomalen Stoffwechsel humaner retinaler Pigmentepithel- (RPE-)Zellen. Als weiterer moglicher Schadigungsmechanismus wurde hier das phototoxische Potenzial des Retinoidanteils untersucht. Methode. Die A2-E-Akkumulation wurde durch Fluoreszenzmikroskopie und FACS-Analyse erfasst. Mittels Acridine-Orange-Farbungen wurden die lysosomale Stabilitat und der intralysosomale pH untersucht. A2-E beladene RPE-Zellen sowie Kontrollen ohne A2-E wurden kurzwelligem sichtbaren Licht (400–500 nm) ausgesetzt, und die Uberlebensrate der RPE-Zellen wurde gemessen. Ergebnisse. Fluoreszenzmikroskopie und FACS-Analyse zeigten eine intralysosomale Akkumulation von A2-E. Acridine-Orange-Farbungen bestatigten die lysosomale Speicherung und pH-Steigerung nach A2-E-Applikation. A2-E beladene und belichtete RPE-Zellen zeigten eine signifikante Reduktion der Uberlebensrate im Vergleich zu ausschlieslich belichteten oder A2-E beladenen Zellkulturen. A2-E-Exposition fuhrt zu einem Verlust lysosomaler Stabilitat. Schlussfolgerung. A2-E schadigt RPE-Zellen offensichtlich durch eine Vielzahl von Mechanismen einschlieslich einer Hemmung des lysosomalen Abbaus, Verlust der Integritat von Zellmembranen und Phototoxizitat. Diese Mechanismen konnten bei der Pathogenese retinaler Erkrankungen eine Rolle spielen, die mit exzessiver Lipofuszinakkumulation im RPE einhergehen einschlieslich altersabhangiger Makuladegeneration, Morbus Best und Morbus Stargardt.

Hintergrund: Lipofuszin akkumuliert in postmitotischen retinalen Pigmentepithelzellen altersabhangig und im Rahmen zahlreicher Netzhauterkrankungen. Um einen moglichen Effekt von Lipofuszinbestandteilen auf die Zellfunktion und damit die pathophysiologische Bedeutung in vitro zu untersuchen, ist eine spezifische Beladung des lysosomalen Kompartiments erforderlich. Wir untersuchten, inwieweit dies mit einer Ankopplung des Lipofuszin-Fluorophors N-Retinyliden-N-Retinylathanolamin (A2-E) an LDL-Partikel erreicht werden kann. Material und Methode: A2-E wurde aus Retinal und Athanolamin synthetisiert und in einen Komplex mit LDL-Partikeln gebracht. Primare humane RPE-Zellkulturen wurden mit dem A2-E/LDL-Komplex uber 4 Wochen inkubiert. Anschliesend wurden die Zellen mittels Stickstoff-Kavitation eroffnet und durch Ultrazentrifugation fraktioniert. Der postnukleare Uberstand wurde hinsichtlich der Markerenzymaktivitaten der verschiedenen zellularen Kompartimente analysiert. Ergebnisse: Im Percoll-Gradienten zeigten sich ubereinstimmende Peaks des lysosomalen Markerenzyms s-Hexosaminidase und der relativen A2-E-Fluoreszenz. Ein Anteil von A2-E korrespondierte mit der Verteilung der Phosphodiesteraseaktivitat als Hinweis fur eine in der Plasmamembran verbliebene geringe Menge von A2-E. Das lysosomale Markerenzym s-Hexosaminidase trat nicht in das zytosolische Kompartiment aus. Schlusfolgerung: Durch die Kopplung von A2-E an LDL-Partikel ist eine effektive Einschleusung eines Retinoid-Bestandteils von Lipofuszin in das lysosomale Kompartiment humaner RPE-Zellen moglich. Das Verfahren kann somit fur Untersuchungen zur Wirkung von A2-E auf pigmentepitheliale Zellfunktionen und damit fur die Klarung der Frage hilfreich sein, inwieweit der Lipofuszinakkumulation im retinalen Pigmentepithel eine pathophysiologische Bedeutung bei Netzhauterkrankungen, die mit einer exzessiven Lipofuszinakkumulation einhergehen, einschlieslich der altersabhangigen Makuladegeneration, zukommt. Die Befunde sprechen gegen eine Detergentienwirkung von A2-E auf die lysosomale Membran.

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

BACKGROUND: A bilateral monofocal detachment of the pigment epithelium (RPE) without any signs of ARMD or other retinal pathology represents a therapeutic challenge. PATIENT: An otherwise healthy 51-year-old woman presented first with a monocular, later a binocular decrease of visual acuity and metamorphopsia. Optical coherence tomography (OCT) showed a bilateral dome-shaped detachment of the RPE. Pooling beneath the detachment was documented using fluorescein angiography. A choroidal neovascularisation could be excluded by means of indocyanine green angiography (ICG). Due to the binocular decrease of visual acuity from 20/20 to 20/63 on the right eye and 20/100 on the left eye in spite of intensive systemic acetazolamide therapy both eyes were treated with photodynamic therapy (PDT) using verteporfin. During monthly controls, the visual acuity increased up to 20/20 on the right eye and 20/25 on the left eye. Metamorphopsia was also reduced. CONCLUSION: An idiopathic detachment of the pigment epithelium can effectively be treated using PDT whereas the underlying pathology remains unclear.

Purpose To visualize retinal pigment epithelial cells in vivo by fundus autofluorescence imaging using a confocal scanning laser ophthalmoscope. Design Experimental study and observational case report. Methods Digital in vivo autofluorescence images were recorded with a confocal scanning laser ophthalmoscope (excitation, 488 nm; emission, >500 nm) and compared with confocal scanning laser ophthalmoscope and fluorescence microscopic recordings from human donor eyes. Results A uniform pattern of the polygonal retinal pigment epithelial cell layer was visualized in vivo outside of absorbing retinal vessels and macular pigment. Autofluorescence intensities of individual cells showed marked variation. The pattern corresponded to in vitro findings. Visualization is based on the topographic distribution of autofluorescent lipofuscin granules and melanin granules in apical retinal pigment epithelium cytoplasm. Conclusions High-resolution autofluorescence imaging may be useful to determine morphologic and lipofuscin-dependent alterations in retinal diseases and may be applicable for monitoring effects of therapeutic interventions targeting the retinal pigment epithelium.

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.

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.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.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 ofor=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 ofor=3 lines. No intraocular or systemic side effects were observed.In this as yet largest series of patients with mCNV treated with intravitreal bevacizumab, the treatment seemed to be effective and safe.

We present an optical coherence tomography (OCT)-based individual reinjection procedure for bevacizumab treatment in neovascular age-related macular degeneration (ARMD).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.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 ofor=3 lines.OCT-based reinjections of bevacizumab in neovascular ARMD reduce the number of injections and lead to anatomic and functional retinal stabilisation.

An intact retinal pigment epithelium (RPE) represents an essential condition for the visual process. This post-mitotic RPE monolayer combines different functions such as degradation of photoreceptor outer segments, vitamin A cycle, support of retinal metabolism and maintenance of the outer blood-retina barrier. As a consequence of excessive metabolism, high oxygen levels, exposition to light of short wave length and ensuing radical formation, the RPE is highly dependent on protective systems. In spite of differentiated defence mechanisms, aging processes cause cumulative RPE damage, representing a major component of age-related macular degeneration (AMD), the leading cause of irreversible severe vision loss in people over 50 years old. A better understanding of the underlying pathophysiology will help to develop new prophylactic options which is becoming more and more important with increasing life expectancy.

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.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.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.OCT-guided reinjection leads to anatomic and functional stabilization or improvement even if transient recurrence of ME occurs.

To report on the efficacy of intravitreal bevacizumab as off-label therapy in different angiographic subtypes in neovascular age-related macular degeneration (AMD).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.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.Intravitreal bevacizumab treatment is efficacious in all angiographic CNV subtypes and leads to reduction of macular edema and stabilization or improvement in visual acuity.

Gorlin Goltz syndrome is a rare, autosomal dominant inherited disease that is characterised by multiple basal cell carcinomas (BCC) including the periorbital region and eye lids. We report a severe infantile manifestation with lid involvement treated by photodynamic therapy (PDT).A 13-year-old boy with Gorlin Goltz syndrome presented with multiple confluent BCC on both eye lids and the skin of neck and trunk. Multiple bilateral periorbital confluent and surgically not removable BCC were treated by topical PDT.Numerous superficial BCC were successfully treated by photodynamic therapy with remarkable cosmetic results.In cases of numerous confluent and surgically not removable BCC, PDT represents an effective therapy. Frequent monitoring is necessary to maintain the clinical outcome.

A bilateral monofocal detachment of the pigment epithelium (RPE) without any signs of ARMD or other retinal pathology represents a therapeutic challenge.An otherwise healthy 51-year-old woman presented first with a monocular, later a binocular decrease of visual acuity and metamorphopsia. Optical coherence tomography (OCT) showed a bilateral dome-shaped detachment of the RPE. Pooling beneath the detachment was documented using fluorescein angiography. A choroidal neovascularisation could be excluded by means of indocyanine green angiography (ICG). Due to the binocular decrease of visual acuity from 20/20 to 20/63 on the right eye and 20/100 on the left eye in spite of intensive systemic acetazolamide therapy both eyes were treated with photodynamic therapy (PDT) using verteporfin. During monthly controls, the visual acuity increased up to 20/20 on the right eye and 20/25 on the left eye. Metamorphopsia was also reduced.An idiopathic detachment of the pigment epithelium can effectively be treated using PDT whereas the underlying pathology remains unclear.

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.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.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.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.

In people over 50, age-related macular degeneration (ARMD) has become the most common cause for severe visual loss and legal blindness in all industrialized nations. Currently, there is no effective treatment for the majority of patients. To develop new and effective modes of therapy, understanding of the molecular basis of the disease in mandatory. However, the pathogenesis of ARMD is still poorly understood. Several lines of evidence suggest that aging changes of the retinal pigment epithelium (RPE), in particular the accumulation of autofluorescent lipofuscin granules in the lysosomal compartment of postmitotic RPE cells, play a key role in the pathogenesis of the disease. Recent studies indicate that lipidic compounds of lipofuscin, represented by the retinoid A2-E, and protein damage by lipid peroxidation products, in particular malondialdehyde and 4-hydroxynonenal, induce lysosomal dysfunction and lipofuscinogenesis in the RPE. The possible mechanisms underlying this lysosomal dysfunction and the resulting adverse effects on overall RPE function are discussed.

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.

Sowohl experimentelle als auch klinische Befunde weisen darauf hin, dass ionisierende Strahlen mature und neu entstehende Gefase beeinflussen konnen. Auf dieser Grundlage wurde die Strahlentherapie bei den Formen der AMD erwogen, die mit Gefasneubildungen einhergehen, also Spatstadien der AMD mit choroidalen Neovaskularisationen. Dabei wurde davon ausgegangen, dass die Wirkung der Bestrahlung auf die verschiedenen okularen Gewebeanteile sehr unterschiedlich ist. Entsprechend der Differenzierung der einzelnen Zellsysteme weisen proliferierende Endothelzellen eine sehr hohe Strahlensensibilitat auf.

Purpose Lipofuscin (LF) accumulation in the retinal pigment epithelium (RPE) is associated with age and various retinal diseases. Toxic LF compounds may interfere with normal RPE function. Oxidative modification of proteins was determined in LF granules from human eyes. Methods LF was isolated from the RPE-choroid complex of 10 pairs of donor eyes by gradient ultracentrifugation. Protein compounds were separated by two-dimensional (2-D) gel electrophoresis and screened by Western blot analysis for lipid peroxidation- or glucoxidation-induced damage-in particular, by malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGEs). Identity of the immunostained proteins was revealed using 2-D software for comparison of the spot position with Coomassie-stained 2-D gels of the same samples. Results By comparing the results taken from the authors' previous proteome analysis of RPE LF with an immunoblot analysis of the same samples, this study shows that a variety of LF-associated proteins were damaged by aberrant covalent modifications of MDA, 4-HNE, and AGEs. Several proteins were altered by two or three different modification types. Modified mitochondrial proteins indicated that autophagy of altered proteins also contributed to lipofuscin formation. Conclusions The identification of lipid peroxidation and glucoxidation products in proteinaceous LF components in human RPE supports the hypothesis that these compounds are involved in lipofuscinogenesis and may contribute to the cytotoxic effects of LF in retinal diseases such as age-related macular degeneration and Stargardt disease. Their identification may help to identify potential future treatment targets.

Background/aims: To document the natural history and to assess the efficacy of interventional therapies in neovascular age related macular degeneration (AMD), an accurate and reproducible method is required for analysis of consecutive fluorescence angiograms. The development and evaluation of an image analysis software for this purpose is described here. It allows for the quantitative analysis of changes in CNV and/or leakage area over time. Methods: In digitised angiograms, a mouse driven arrow was used to delineate the CNV border. The ratio of the CNV area to the square of the distance between two vessels was automatically calculated by pixel count to compensate for variation in image sizes at different examination times. These results were directly transferred and stored in a database. To assess reproducibility, CNV areas in 20 patients with occult and 20 patients with classic CNV were determined independently by two readers. Results: There was only marginal variability between observers with this method: the mean deviation was 0.01 pixels for classic CNV (95% CI −0.17 to +0.15, SD 0.35) and 0.55 pixels for occult CNV (95% CI −1.06 to −0.04, SD 1.14). Conclusions: This practical PC based method allows for quantification of angiographic features such as CNV size in early frames and area of leakage in late frames. Limitations include non-readily defined borders in angiograms of poor image quality or indistinct borders of the hyperfluorescent areas of interest. The software is applicable to future clinical trials where the analysis of neovascular complex changes is required, for example, following therapeutic intervention.

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.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.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.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.