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Abstract Retinopathy of prematurity (ROP) is a severe retinal disease in premature infants characterized by pathological neovascularization, obliteration of retinal vessels and increased vessel tortuosity. Currently, there are no completely satisfactory treatments for ROP. Pigment epithelium‐derived factor (PEDF), a potent inhibitor of angiogenesis, appears late in gestation and its deficiency may be linked to development of ROP. This study investigates the preclinical efficacy of PEDF protein alone or in combination with VEGF antagonists for treating ROP. The safety of PEDF protein in the rat eye was assessed using functional in vivo measurements and histology. The efficacy of intravitreal injections (IVI) of various treatments was evaluated in a rat oxygen‐induced retinopathy (OIR) model using in vivo imaging and flatmount analyses. No functional or histological side‐effects were found in rat eyes after intravitreal PEDF protein injection. PEDF protein alone or combined with anti‐VEGF drugs significantly reduced pathological neovascularization and vessel obliteration, comparable to the effects of anti‐VEGF drugs alone. Regarding arterial tortuosity, treatment with a combination of PEDF, and VEGF antagonist was more effective than treatment with anti‐VEGF alone. IVI of PEDF protein is safe. PEDF protein alone or combined with VEGF antagonists shows similar efficacy in reducing pathological neovascularization and vessel obliteration as anti‐VEGF agents. Furthermore, only treatments involving PEDF protein, alone or with VEGF antagonists, significantly improved the quality of retinal vasculature. Thus, PEDF protein alone or combined with anti‐VEGF agents presents a promising alternative to current anti‐VEGF treatments for ROP.

Abstract Lipofuscin is a representative biomarker of aging that is generated naturally over time. Remofuscin (soraprazan) improves age-related eye diseases by removing lipofuscin from retinal pigment epithelium (RPE) cells. In this study, the effect of remofuscin on longevity in Caenorhabditis elegans and the underlying mechanism were investigated. The results showed that remofuscin significantly (p

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

Abstract 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.

Vascular endothelial growth factor (VEGF) is a crucial stimulator for choroidal neovascularization (CNV). Our aim was to develop a reproducible and valid treatment-naive quiescent CNV (i.e. without signs of exudation and with normal visual acuity) rat model by subretinal injection of an adeno-associated virus (AAV)-VEGFA165 vector. The CNV development was longitudinally followed up in vivo by scanning laser ophthalmoscopy/optical coherence tomography, fluorescein and Indocyanine Green angiographies and ex vivo by electron microscopy (EM) and immunohistochemistry. In total, 57 eyes were analysed. In vivo, a quiescent CNV was observed in 93% of the eyes 6 weeks post-transduction. In EM, CNV vessels with few fenestrations, multi-layered basement membranes and bifurcation of endothelial cells were observed sharing the human CNV features. Human VEGF overexpression, multi-layered retinal pigment epithelium (RPE) (RPE65) and macrophages/activated microglia (Iba1) were also detected. In addition, 19 CNV eyes were treated for up to 3 weeks with bevacizumab. The retinal and CNV lesion thickness decreased significantly in bevacizumab-treated CNV eyes compared with untreated CNV eyes 1 week after the treatment. In conclusion, our experimental CNV resembles those seen in patients suffering from treatment-naive quiescent CNV in wet age-related macular degeneration (AMD), and responds to short-term treatment with bevacizumab. Our new model can, therefore, be used to test the long-term effect of new drugs targeting CNV under precisely-defined conditions.

The data presented in this article are related to the research paper entitled “The anatomy of the foveola reinvestigated” (Tschulakow et al., 2018) [1]. Here we show the original aligned serial sections through the foveal centre of monkeys at different planes of section and 3 D models of central foveal cells. Keywords: Müller glial cells, Fovea, Cone receptors, Foveola, 3D model, Umbo, Stiles-Crawford effect

Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining. Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patient's tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies.

AIM: To compare the effects on the retina inner segment and retinal pigment epithelium(RPE)of intravitreally injecting bevacizumab, ranibizumab and aflibercept into monkey eyes.METHODS: Fourteen healthy cynomolgus monkeys(Macaca fascicularis, aged 3-8y,10 males,4 females)were raised at the Covance Laboratories under standard conditions. The 14 monkeys were grouped into 4 groups. Three of the groups with 4 monkeys each were injected intravitreally with one of the drugs, either bevacizumab, ranibizumab or aflibercept, while the 4th group with 2 monkeys served as a negative control. On 1d and 7d of injection, 2 monkeys from each drug treatment group were sacrificed under general anaesthesia and the 4 eyes were enucleated. All the enucleated eyes were fixed in formalin, embedded in paraffin wax, cut into 4.0 μm sections and deparaffinized according to standard procedures. Image-Pro Plus was used for all the photos to measure the content of vascular endothelial growth factor(VEGF)in the inner segment and RPE. The ANOVA test from JMP10.0 statistical program was used to evaluate the results.RESULTS: Retinal sections were checked for their anti-VEGF immune reactivity. The untreated control samples had the highest level of VEGF in the RPE and inner segment. All of these three drugs can reduce the level of VEGF in the RPE and inner segment, but Avastin seems to be more effective than Eylea in this regard. Lucentis treatment at 1d seems to be more effective than Eylea at VEGF 1d. But at 7d, both Lucentis and Eylea have the same effect on reducing VEGF expression level in the RPE and inner segment.CONCLUSION: All of these three drugs can reduce the level of VEGF in the RPE and inner segment.

Background Stargardt disease (SD) is characterized by the accumulation of the age-pigment lipofuscin in the retinal pigment epithelium (RPE) and subsequent neuroretinal degeneration. The disease leads to vision loss early in life. Here, we investigate age-dependent ultrastructural changes in three SD mouse models: albino Abca4-/- and pigmented Abca4-/- and Abca4-/-.Rdh8-/- mice. Since we found indications for oxidative stress primarily in albino SD mice, we tested RPE melanin for its antioxidative capabilities. Methods SD mouse eyes were investigated by light, fluorescence and electron microscopy and were compared to the respective albino and pigmented wild type mice and to a human donor SD eye. To confirm the role of RPE melanin in scavenging oxidative stress, melanin from S. officinalis as a standard and porcine RPE were tested for their capability to quench superoxide anions. Results Histological alterations indicative of oxidative stress and/or lysosomal dysfunction were present in albino Abca4-/- and Abca4-/-.Rdh8-/- mice. Retinal damage, such as inner segment rupture and pyknotic or free photoreceptor nuclei in the subretinal space and RPE vacuolization were exclusively found in albino Abca4-/- mice. Shortened and disorganized photoreceptor outer segments and dead RPE cells were found in albino Abca4-/- and Abca4-/-.Rdh8-/- mice, with earlier onset in albino Abca4-/- mice. Undegraded phagosomes and lipofuscin accumulation were present in the RPE of all three SD strains, but numbers were highest in Abca4-/-.Rdh8-/- mice. Lipofuscin morphology differed between SD strains: (melano-)lipofuscin granules in pigmented Abca4-/- mice had a homogenous electron density and sharp demarcations, while lipofuscin in albino Abca4-/- mice had a flocculent electron density and often lacked a surrounding membrane, indicating loss of lysosomal integrity. Young Abca4-/-.Rdh8-/- mice showed (melano-)lipofuscin granules with homogenous electron density, while in aged animals granules with flocculent electron density predominated. Both strains of pigmented SD mice had melanolipofuscin clusters as found in the human SD eye. Like melanin from S. officinalis, porcine RPE melanin can also quench superoxide anions. Discussion The presented pathologies in albino Abca4-/- and Abca4-/-.Rdh8-/- mice suggest oxidative stress and/or lysosomal dysfunction within the RPE. Since albino Abca4-/- mice have the earliest onset and severest damage and as absence of melanin and also melanin turnover with age are known to diminish RPEs anti-oxidative properties, we assume that RPE melanin plays a role in SD related damages. A lack of pathology in pigmented Abca4-/- mice due to lower stress levels as compared to the Abca4-/-.Rdh8-/- mice underlines this hypothesis. It is also supported by the finding that RPE melanin can quench superoxide anions. We therefore suppose that RPE melanin is important in retinal health and we discuss its role as an oxidative stress scavenger.

Objective In the foveola of the eye, photoreceptors and Müller cells with a unique morphology have been described, but little is known about their 3D structure and orientation. Considering that there is an angle-dependent change in the foveolar photoreceptor response for the same light beam, known as the Stiles Crawford Effect of the first kind (SCE I), which is still not fully understood, a detailed analysis of the anatomy of the foveolar cells might help to clarify this phenomenon. Methods Serial semithin and ultrathin sections, and focused ion beam (FIB) tomography were prepared from 32 foveolae from monkeys (Macaca fascicularis) and humans. Foveolae were also analyzed under the electron microscope. Serial sections and FIB analysis were then used to construct 3D models of central Müller and photoreceptor cells. In addition, we measured the transmission of collimated light under the light microscope at different angles after it had passed through human foveae from flat mounted isolated retinae. Results In monkeys, outer segments of central foveolar cones are twice as long as those from parafoveal cones and do not run completely parallel to the incident light. Unique Müller cells are present in the central foveolae (area of 200 µm in diameter) of humans and monkeys. Light entering the fovea center, which is composed only of cones and Müller cells, at an angle of 0° causes a very bright spot after passing through this area. However, when the angle of the light beam is changed to 10°, less light is measured after transpasssing through the retina, the foveolar center becomes darker and the SCE-like phenomenon is directly visible. Measurements of the intensities of light transmission through the central foveola for the incident angles 0 and 10° resemble the relative luminance efficiency for narrow light bundles as a function of the location where the beam enters the pupil as reported by Stiles and Crawford. The effect persisted after carefully brushing away the outer segments. Conclusion We show that unique cones and Müller cells with light fibre-like properties are present in the center of the fovea. These unique Müller cells cause an angle dependent, SCE-like drop in the intensity of light guided through the foveola. Outer segments from the foveolar cones of monkeys are not straight.

Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports. Here, we assess the value of OCT reflectivity profiles as a basis for a quantitative characterization of the retinal status in a cross-species comparative study.Spectral-Domain Optical Coherence Tomography (OCT), confocal Scanning-Laser Ophthalmoscopy (SLO), and Fluorescein Angiography (FA) were performed in mice (Mus musculus), gerbils (Gerbillus perpadillus), and cynomolgus monkeys (Macaca fascicularis) using the Heidelberg Engineering Spectralis system, and additional SLOs and FAs were obtained with the HRA I (same manufacturer). Reflectivity profiles were extracted from 8-bit greyscale OCT images using the ImageJ software package (http://rsb.info.nih.gov/ij/).Reflectivity profiles obtained from OCT scans of all three animal species correlated well with ex vivo histomorphometric data. Each of the retinal layers showed a typical pattern that varied in relative size and degree of reflectivity across species. In general, plexiform layers showed a higher level of reflectivity than nuclear layers. A comparison of reflectivity profiles from specialized retinal regions (e.g. visual streak in gerbils, fovea in non-human primates) with respective regions of human retina revealed multiple similarities. In a model of Retinitis Pigmentosa (RP), the value of reflectivity profiles for the follow-up of therapeutic interventions was demonstrated.OCT reflectivity profiles provide a detailed, quantitative description of retinal layers and structures including specialized retinal regions. Our results highlight the potential of this approach in the long-term follow-up of therapeutic strategies.

It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm.Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased.Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.

It is textbook knowledge that human infective forms of Trypanosoma brucei, the causative agent of sleeping sickness, enter the brain across the blood-brain barrier after an initial phase of weeks (rhodesiense) or months (gambiense) in blood. Based on our results using an animal model, both statements seem questionable. As we and others have shown, the first infection relevant crossing of the blood brain border occurs via the choroid plexus, i.e. via the blood-CSF barrier. In addition, counting trypanosomes in blood-free CSF obtained by an atlanto-occipital access revealed a cyclical infection in CSF that was directly correlated to the trypanosome density in blood infection. We also obtained conclusive evidence of organ infiltration, since parasites were detected in tissues outside the blood vessels in heart, spleen, liver, eye, testis, epididymis, and especially between the cell layers of the pia mater including the Virchow-Robin space. Interestingly, in all organs except pia mater, heart and testis, trypanosomes showed either a more or less degraded appearance of cell integrity by loss of the surface coat (VSG), loss of the microtubular cytoskeleton and loss of the intracellular content, or where taken up by phagocytes and degraded intracellularly within lysosomes. This is also true for trypanosomes placed intrathecally into the brain parenchyma using a stereotactic device. We propose a different model of brain infection that is in accordance with our observations and with well-established facts about the development of sleeping sickness.

BACKGROUND: Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet. METHODOLOGY/PRINCIPAL FINDINGS: Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch's membrane of ZD-LE rats varied between 0.4-3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch's membrane or even inside it. CONCLUSIONS/SIGNIFICANCE: In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch's membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch's membrane.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.