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Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is a hallmark of aging and is associated with retinal degeneration encountered in age-related macular degeneration (AMD) and Stargardt disease (SD). Currently, treatment for lipofuscin-induced retinal degeneration is unavailable. Here, we report that Remofuscin (INN: soraprazan, a tetrahydropyridoether small molecule) reverses lipofuscin accumulation in aged primary human RPE cells and is non-cytotoxic in aged SD mouse RPE cells in vitro. In addition, we show that the removal of lipofuscin after a single intravitreal injection of Remofuscin results in a rescue from retinal degeneration in a mouse model of advanced SD which is even accompanied by an amelioration of the retinal dysfunction. Finally, we demonstrate that the mechanism causing lipofuscinolysis may involve the reactive oxygen species generated via the presence of Remofuscin. These data suggest a possible therapeutic approach to untreatable lipofuscin-mediated diseases like AMD, SD and lipofuscinopathies in neurodegenerative diseases.

Ocular ischemia/hypoxia is a severe problem in ophthalmology that can cause vision impairment and blindness. However, little is known about the changes occurring in the existing fully formed choroidal blood vessels. We developed a new whole organ culture model for ischemia/hypoxia in rat eyes and investigate the effects of pigment epithelium derived factor (PEDF) protein on the eye tissues. The concentration of oxygen within the vitreous was measured in the enucleated rat eyes and living rats. Then, ischemia was mimicked by incubating the freshly enucleated eyes in medium at 4°C for 14 h. Eyes were fixed immediately after enucleation or were intravitreally injected with PEDF protein or with vehicle before incubation. After incubation, light and electron microscopy (EM) as well as Tunel staining was performed. In the living rats, the intravitreal oxygen concentration was on average at 16.4% of the oxygen concentration in the air and did not change throughout the experiment whereas it was ca. 28% at the beginning of the experiment and gradually decreased over time in the enucleated eyes. EM analysis revealed that the shape of the choriocapillaris changed dramatically after 14 h incubation in the enucleated eyes. The endothelial cells made filopodia-like projections into the vessel lumen. They appeared identical to the labyrinth capillaries found in surgically extracted choroidal neovascular membranes from patients with wet age-related macular degeneration (AMD). These filopodia-like projections nearly closed the vessel lumen and showed open gaps between neighboring endothelial cells. PEDF significantly inhibited labyrinth capillary formation and kept the capillary lumen open. The number of TUNEL-positive ganglion cells and inner nuclear layer cells was significantly reduced in the PEDF-treated eyes compared to the vehicle-treated eyes. The structural changes in the chroidal vessels observed under ischemia/hypoxia conditions can mimic early changes in the process of pathological angiogenesis as observed in wet AMD patients. This new model can be used to investigate short-term drug effects on the choriocapillaris after ischemia/hypoxia and it highlighted the potential of PEDF as a promising candidate for treating wet AMD.

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.

Background: Fundus autofluorescence is a non-invasive imaging technique in ophthalmology. Conventionally, short-wavelength autofluorescence (SW-AF) is used for detection of lipofuscin, a byproduct of the visual cycle which accumulates with age or disease in the retinal pigment epithelium (RPE). Furthermore, near-infrared autofluorescence (NIR-AF) is used as a marker for RPE and choroidal melanin, but contribution of lipofuscin to the NIR-AF signal is unclear. Methods: We employed fluorescence microscopy to investigate NIR-AF properties of melanosomes, lipofuscin and melanolipofuscin granules in histologic sections of wildtype and Abca4−/− mouse eyes, the latter having increased lipofuscin, as well as aged human donor eyes. Differentiation between these pigments was verified by analytical electron microscopy. To investigate the influence of oxidative and photic stress we used an in vitro model with isolated ocular melanosomes and an in vivo phototoxicity mouse model. Findings: We show that NIR-AF is not an intrinsic property of melanin, but rather increases with age and after photic or oxidative stress in mice and isolated melanosomes. Furthermore, when lipofuscin levels are high, lipofuscin granules also show NIR-AF, as confirmed by correlative fluorescence and electron microscopy in human tissue. However, lipofuscin in albino Abca4−/− mice lacks NIR-AF signals. Interpretation: We suggest that NIR-AF is derived from melanin degradation products that accumulate with time in lipofuscin granules. These findings can help to improve the interpretation of patient fundus autofluorescence data. Funding: This work was supported by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft and Chinese Scholarship Council. Major instrumentation used in this work was supported by Deutsche Forschungsgemeinschaft, the European Fund for Regional Development and the state of Baden-Württemberg. Keywords: Melanin, Lipofuscin, Melanolipofuscin, Oxidative stress, Aging

Age‐related macular degeneration (AMD) is the leading cause of blindness in older people in the developed world while Stargardt's disease (SD) is a juvenile macular degeneration and an orphan disease. Both diseases are untreatable and are marked by accumulation of lipofuscin advancing to progressive deterioration of the retinal pigment epithelium (RPE) and retina and subsequent vision loss till blindness. We discovered that a small molecule belonging to the tetrahydropyridoether class of compounds, soraprazan renamed remofuscin, is able to remove existing lipofuscin from the RPE. This study investigated the drug penetration, distribution, and elimination into the eyes of a mouse model for increased lipofuscinogenesis, following a single intravitreal injection. We measured the time course of concentrations of remofuscin in different eye tissues using high‐performance liquid chromatography combined with mass spectroscopy (HPLC‐MS). We also visualized the penetration and distribution of 3H‐remofuscin in eye sections up to 20 weeks post‐injection using transmission electron microscopic (TEM) autoradiography. The distribution of silver grains revealed that remofuscin accumulated specifically in the RPE by binding to the RPE pigments (melanin, lipofuscin and melanolipofuscin) and that it was still detected after 20 weeks. Importantly, the melanosomes in choroidal melanocytes only rarely bind remofuscin emphasizing its potential to serve as an active ingredient in the RPE for the treatment of SD and dry AMD. In addition, our study highlights the importance of electron microscopic autoradiography as it is the only method able to show drug binding with a high intracellular resolution., TEM autoradiography shows: a depot effect of remofuscin that could be relevant for a local application of the drug. The RPE specificity of the drug. Only TEM autoradiography allows the investigation of drug binding with high intracellular resolution.

Background Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho- L -tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xeno graft and a genetic orthotopic Rb mouse model. Methods B6;129-Rb1tm3Tyj/J (Rb +/− ) and Y79-Rb cell- xeno grafted nude mice were fractionated external beam irradiated (15 fractions of 5 Gy 6 MV photons during 3 weeks) with or without pTyr pre-treatment (100 mg/kg BW, 16 h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro . Results In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb +/− mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xeno grafted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. Conclusions In Rb +/− mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb +/− mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xeno graft model, pTyr treatment reduced the control of Rb tumours.

Anti-vascular endothelial growth factor (VEGF) drugs are used to treat neovascular eye diseases. Some of these drugs contain Fc fragments (Fc), but it is unknown how their mode of action is influenced by Fc. Therefore, this study investigated the effects of Fc on rat eyes after intravitreal injection. Eighteen Long–Evans rats were intravitreally injected with sterile, biotin-labeled rat Fc (9.1 μg in 5 μl PBS). For control, 5 μl PBS was injected in another nine rats. Animals were sacrificed between 1 and 3 days (group 1), 7 days (group 2), and 14 days (group 3) after injection. The right eyes were examined by electron microscopy (EM). The left eyes were stained by immunohistochemistry to investigate the distribution of Fc and the presence of macrophages. After 1 day, Fc had penetrated into the anterior chamber and the retina up to the inner nuclear layer, and was located especially in retinal vessels. High numbers of infiltrating cells were present within the vitreous, around the ciliary body, anterior chamber and inside the retina 1–3 days after Fc injection (p

To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.