Optical coherence tomography angiography suggests choriocapillaris perfusion deficit as etiology of acute macular neuroretinopathy.

PURPOSE: Acute macular neuroretinopathy (AMN) can cause sudden-onset and permanent scotoma in healthy young patients. Analysis of optical coherence tomography (OCT) and OCT angiography (OCTA) of AMN patients may provide insights into disease mechanism. METHODS: We conducted a retrospective study of consecutive SARS-Cov-2-related AMN patients that presented in our clinic between Jan 1st, 2022, and April 30th, 2023, within 30 days of symptom onset. Retinal vessel area density (VAD) of AMN lesions in OCTA was quantified and compared to an adjacent tissue control (ATC). This quantification was performed for the superficial vascular plexus (SVP), the intermediate capillary plexus (ICP), the deep capillary plexus (DCP), the choriocapillaris (CC), and choroid. Furthermore, en face OCT images were analyzed. RESULTS: Nine AMN patients were identified, 6 of these (4 female, 2 male, average age 25 years) fulfilled the inclusion criteria and were included into this study. Average time from symptom onset to OCTA was 14.3 days. No VAD differences between AMN and adjacent tissue were found in either retinal layer (SVP, ICP, DCP). In contrast, VAD in CC was reduced by 27% against the ATC (p = 0.007) and choroidal VAD was reduced by 41% (p = 0.017). Further analysis of en face OCT could show that the pathognomonic infrared hyporeflectivity in AMN is caused by photoreceptor alterations rather than changes in the inner retinal layers. CONCLUSIONS: Our data suggests that a perfusion deficit in the choroidal layers is responsible for AMN rather than in the DCP, which is the predominant hypothesis in current literature.

LASER FLARE PHOTOMETRY IN PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT: An Evaluation of 2,487 Cases.

PURPOSE: Exploratory analysis associated with the prospective, multicenter, randomized PRIVENT trial. To characterize the associations between laser flare photometry and anatomical and epidemiological features of rhegmatogenous retinal detachment (RRD). METHODS: The authors measured laser flare values of all 3,048 prescreened patients excluding those with comorbidities. A mixed regression analysis evaluated the strength of the influencing factors like age, sex, lens status, and presence and extent of RRD on laser flare. RESULTS: Rhegmatogenous retinal detachment was more frequent in men (65.8%) than in women (34.2%, P < 0.001) and in right (52%) than in left eyes (48%, P = 0.045). Phakic RRD affected less quadrants and was less likely to be associated with macula-off status than pseudophakic RRD (48.4% vs. 58.0% macula off, 23% vs. 31% ≥3 quadrants, P < 0.001). Laser flare of affected eyes was significantly higher compared with fellow eyes (12.6 ± 15.2 vs. 8.3 ± 7.4 pc/ms, P < 0.001). The factors age, sex, lens status, presence of RRD, and the number of quadrants affected were independent influencing factors on laser flare. R 2 was 0.145 for phakic and 0.094 for pseudophakic eyes. CONCLUSION: The results indicate that there may be more factors affecting laser flare than previously assumed. This might limit flare as predictive value for PVR and retinal redetachment.

Effects of Fucoidans on Activated Retinal Microglia.

Sulfated marine polysaccharides, so-called fucoidans, have been shown to exhibit anti-inflammatory and immunomodulatory activities in retinal pigment epithelium (RPE). In this study, we tested the effects of different fucoidans (and of fucoidan-treated RPE cells) on retinal microglia to investigate whether its anti-inflammatory effect can be extrapolated to the innate immune cells of the retina. In addition, we tested whether fucoidan treatment influenced the anti-inflammatory effect of RPE cells on retinal microglia. Three fucoidans were tested (FVs from Fucus vesiculosus, Fuc1 and FucBB04 from Laminaria hyperborea) as well as the supernatant of primary porcine RPE treated with fucoidans for their effects on inflammatory activated (using lipopolysaccharide, LPS) microglia cell line SIM-A9 and primary porcine retinal microglia. Cell viability was detected with a tetrazolium assay (MTT), and morphology by Coomassie staining. Secretion of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1ß) and interleukin 8 (IL8) was detected with ELISA, gene expression (NOS2 (Nitric oxide synthase 2), and CXCL8 (IL8)) with qPCR. Phagocytosis was detected with a fluorescence assay. FucBB04 and FVs slightly reduced the viability of SIM-A9 and primary microglia, respectively. Treatment with RPE supernatants increased the viability of LPS-treated primary microglia. FVs and FucBB04 reduced the size of LPS-activated primary microglia, indicating an anti-inflammatory phenotype. RPE supernatant reduced the size of LPS-activated SIM-A9 cells. Proinflammatory cytokine secretion and gene expression in SIM-A9, as well as primary microglia, were not significantly affected by fucoidans, but RPE supernatants reduced the secretion of LPS-induced proinflammatory cytokine secretion in SIM-A9 and primary microglia. The phagocytosis ability of primary microglia was reduced by FucBB04. In conclusion, fucoidans exhibited only modest effects on inflammatorily activated microglia by maintaining their cell size under stimulation, while the anti-inflammatory effect of RPE cells on microglia irrespective of fucoidan treatment could be confirmed, stressing the role of RPE in regulating innate immunity in the retina.

Establishment of specific age-related macular degeneration relevant gene expression panels using porcine retinal pigment epithelium for assessing fucoidan bioactivity.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. Important factors in pathogenesis are oxidative stress, inflammation, and, in the wet form of AMD, angiogenesis. Fucoidans, sulfated polysaccharides from brown algae, may have antioxidant, anti-inflammatory, and antiangiogenic effects. In this study, we established specific gene expression panels for inflammation, oxidative stress and angiogenesis in porcine retinal pigment epithelium (RPE), and investigated the effect of fucoidans on gene expression under different noxious agents. METHODS: Primary porcine RPE cells cultured for at least 14 days were used. Using viability assays with tetrazolium bromide and real-time polymerase chain reaction of marker genes, positive controls were established for appropriate concentrations and exposure times of selected noxious agents (lipopolysaccharide (LPS), H2O2, CoCl2). Three different AMD relevant gene panels specific for porcine RPE for inflammation, oxidative stress, and angiogenesis were established, and the influence of fucoidans (mainly Fucus vesiculosus; FV) on gene expression was investigated. RESULTS: The following was shown by gene expression analyses: (1) Inflammation panel: Expression of 18 genes was affected under LPS (three days). Among them, LPS increased genes for interleukin 1 receptor 2, interleukin 8, cyclooxygenase-2 and vascular cell adhesion protein 1 expression which were diminished when FV was present. (2) Oxidative stress panel: Under stimulation of H2O2 (one day) and LPS (one day), expression of a total of 15 genes was affected. LPS induced increase in genes for superoxide dismutase-1, C-X-C motif chemokine 10, and CC chemokine ligand-5 expression was not detected when FV was present. (3) Angiogenesis panel: Under stimulation with CoCl2 (three days) expression of six genes was affected, with the increase of genes for angiopoietin 2, vascular endothelial growth factor receptor-1, and follistatin being diminished when FV was present. CONCLUSION: Three specific gene expression panels for porcine RPE that map genes for three of the major pathological factors of AMD, inflammation, oxidative stress, and angiogenesis, were established. Further, we demonstrated that fucoidans can reduce stress related gene activation in all of these three major pathogenic pathways. This study is another indication that fucoidans can act on different pathomechanisms of AMD simultaneously, which provides further evidence for fucoidans as a possible drug for treatment and prevention of AMD.

Fundus autofluorescence abnormalities can predict fluorescein angiography abnormalities in patients with chronic central serous chorioretinopathy.

PURPOSE: This study is to assess the possible correlation between findings on fundus autofluorescence (FAF) and fluorescein angiography (FA) in patients with chronic central serous chorioretinopathy (cCSC). METHODS: This multicentre retrospective cohort study included 71 cCSC patients (92 eyes) with at least 6 months of follow-up, who had a FAF-FA imaging discrepancy larger than 0.5 optic disc diameters in size in the corresponding areas of hyperfluorescent abnormalities. A comparison was performed between progression in size of areas of hyperautofluorescent retinal pigment epithelium (RPE) abnormalities on FAF (HF-FAF) and the hyperfluorescent areas on FA (HF-FA) at first visit and last visit. The possible correlations were estimated between FAF-FA discrepancy and disease characteristics. RESULTS: The median area of HF-FAF at first visit was 7.48 mm2 (1.41-27.9). The median area of HF-FA at first visit and last visit was 2.40 mm2 (0.02-17.27) and 5.22 mm2 (0.53-25.62), respectively. FAF-FA discrepancy was associated with follow-up duration and the area of HF-FAF at first visit. A mathematical algorithm for grading FAF-FA discrepancy in time was suggested, which predicted the enlargement of hyperfluorescent RPE abnormalities on FA in 82.6% of cases. CONCLUSION: There is a statistically significant relationship between the areas of HF-FAF and HF-FA in cCSC patients with FAF-FA imaging discrepancy at first presentation. Long-term changes in RPE alterations in cCSC on FA can be predicted based on baseline HF-FAF and follow-up duration.

Interaction of High-Molecular Weight Fucoidan from Laminaria hyperborea with Natural Functions of the Retinal Pigment Epithelium.

Fucoidans are polysaccharides and constituents of cell walls of brown algae such as Laminaria hyperborea (LH). They exhibit promising effects regarding age-related macular degeneration (AMD). However, the safety of this compound needs to be assured. The focus of this study lies on influences of an LH fucoidan on the retinal pigment epithelium (RPE). The high-molecular weight LH fucoidan Fuc1 was applied to primary porcine RPE cells, and a tetrazolium (MTT) cell viability assay was conducted. Further tests included a scratch assay to measure wound healing, Western blotting to measure expression of retinal pigment epithelium-specific 65 kDa protein (RPE65), as well as immunofluorescence to measure uptake of opsonized fluorescence beads into RPE cells. Lipopolysaccharide was used to proinflammatorily activate the RPE, and interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion was measured. RPE/choroid cultures were used to assess vascular endothelial growth factor (VEGF) secretion. Real-time polymerase chain reaction (real-time PCR) was performed to detect the gene expression of 91 different genes in a specific porcine RPE gene array. Fuc1 slightly reduced wound healing, but did not influence cell viability, phagocytosis or RPE65 expression. Fuc1 lowered IL-6, IL-8 and VEGF secretion. Furthermore, Fuc1 did not change tested RPE genes. In conclusion, Fuc1 does not impair RPE cellular functions and shows antiangiogenic and anti-inflammatory activities, which indicates its safety and strengthens its suitability concerning ocular diseases.

Comparison of Fucoidans from Saccharina latissima Regarding Age-Related Macular Degeneration Relevant Pathomechanisms in Retinal Pigment Epithelium.

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H2O2. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research.

Intravitreal 5-Fluorouracil and Heparin to Prevent Proliferative Vitreoretinopathy: Results from a Randomized Clinical Trial.

PURPOSE: Proliferative vitreoretinopathy (PVR) is the major cause for surgical failure after primary rhegmatogenous retinal detachment (RRD). So far, no therapy has been proven to prevent PVR. Promising results for 5-fluorouracil (5-FU) and low-molecular weight heparin (LMWH) in high-risk eyes have been reported previously. The objective of this trial was to examine the effect of adjuvant intravitreal therapy with 5-FU and LMWH compared with placebo on incidence of PVR in high-risk patients with primary RRD. DESIGN: Randomized, double-blind, controlled, multicenter, interventional trial with 1 interim analysis. PARTICIPANTS: Patients with RRD who were considered to be at high risk for PVR were included. Risk of PVR was assessed by noninvasive aqueous flare measurement using laser flare photometry. METHODS: Patients were randomized 1:1 to verum (200 mg/ml 5-FU and 5 IU/ml dalteparin) and placebo (balanced salt solution) intravitreally applied during routine pars plana vitrectomy. MAIN OUTCOME MEASURES: Primary end point was the development of PVR grade CP (full-thickness retinal folds or subretinal strands in clock hours located posterior to equator) 1 or higher within 12 weeks after surgery. For grading, an end point committee assessed fundus photographs. Secondary end points included best-corrected visual acuity and redetachment rate. A group sequential design with 1 interim analysis was applied using the O'Brien and Fleming boundaries. Proliferative vitreoretinopathy grade CP incidence was compared using a Mantel-Haenszel test stratified by surgeon. RESULTS: A total of 325 patients in 13 German trial sites had been randomized (verum, n = 163; placebo, n = 162). In study eyes, mean laser flare was 31 ± 26 pc/ms. No significant difference was found in PVR rate. Primary analysis in the modified intention-to-treat population results were: verum 28% vs. placebo 23% (including not assessable cases as failures); odds ratio [OR], 1.25; 95% confidence interval [CI], 0.76-2.08; P = 0.77. Those in the per-protocol population were: 12% vs. 12%; OR, 1.05; 95% CI, 0.47-2.34; P = 0.47. None of the secondary end points showed any significant difference between treatment groups. During the study period, no relevant safety risks were identified. CONCLUSIONS: Rate of PVR did not differ between adjuvant therapy with 5-FU and LMWH and placebo treatment in eyes with RRD.

Influence of carrier materials and coatings on retinal pigment epithelium cultivation and functions.

Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGF-ß) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-ß and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.

Pro-inflammatory activation changes intracellular transport of bevacizumab in the retinal pigment epithelium in vitro.

PURPOSE: Bevacizumab is taken up and transported through the retinal pigment epithelium. Inflammatory signaling may influence this interaction. In the present study, we have investigated the effect of pro-inflammatory stimuli on the uptake, intracellular localization, and transepithelial transport of bevacizumab. METHODS: ARPE-19 cell line or primary porcine RPE cells were treated with clinical relevant concentrations of bevacizumab (250 µg/ml). Pro-inflammatory signaling was induced by TLR-3 agonist polyinosinic:polycytidylic acid (Poly I:C). Viability was investigated with MTT and trypan-blue exclusion assay, and cell number, uptake, and intracellular localization were investigated with immunofluorescence, investigating also actin filaments, the motor protein myosin 7a and lysosomes. Immunofluorescence signals were quantified. Intracellular bevacizumab was additionally detected in Western blot. Barrier function was investigated with transepithelial resistant measurements (TER). The transepithelial transport of bevacizumab and its influence on cytokine (IL-6, IL-8, IL-1ß, TNFα) secretion was investigated with ELISA. RESULTS: Poly I:C in combination with bevacizumab reduced the viability of the cells. Treatment with Poly I:C reduced the uptake of bevacizumab, changed the intensity of the actin filaments, and reduced the colocalization with myosin 7a. In addition, Poly I:C reduced the capacity of RPE cells to transport bevacizumab over the barrier. In addition, bevacizumab reduced the secretion of IL-8 and TNFα after Poly I:C stimulation at selected time points. CONCLUSIONS: Pro-inflammatory activation of RPE cells with TLR-3 agonist Poly I:C changes the interaction of RPE cells with the anti-VEGF compound bevacizumab, reducing its uptake and transport. On the other hand, bevacizumab might influence pro-inflammatory cytokine release. Our data indicate that inflammation may influence the pharmacokinetic of bevacizumab in the retina.

Clinicopathological Correlation of Microbial Keratitis and Ahead: Is There a Corneal Sepsis? / Zur klinisch-pathologischen Korrelation der mikrobiellen Keratitis und darüber hinaus: Gibt es eine korneale Sepsis?

Microbial, infectious keratitis is a relevant indication for penetrating keratoplasty. The requirement for transplantation results in histopathological examination of the entire thickness of the cornea. Although the clinical diagnosis is not always possible to confirm, pathology can support diagnostic evidence of clinical presentation and pathogenesis. This is achieved with multiple methods from cytology, histochemistry, immunohistology, molecular pathology and in rare cases electron microscopy. These allow tissue-based detection of previous and parallel diseases and the responsible pathogens. The failure of satisfactory clinicopathological correlation raises the question whether a suspected pathogen was not ultimately responsible for destroyed corneal tissue. The pathogenesis of keratitis requiring transplantation is not yet completely understood, also on the experimental level. The development of such a keratitis can lead to a clinical symptomatology which can be described as "threatening organ dysfunction", a term used in sepsis research. Considering recent literature, possible correlations between sepsis and microbial keratitis and their relation to histopathology are discussed.

Basolateral activation with TLR agonists induces polarized cytokine release and reduces barrier function in RPE in vitro.

PURPOSE: Systemic inflammation may be of importance in the development of AMD. RPE cells can recognize danger signals with toll-like receptors (TLR) and may react in a pro-inflammatory manner. In this study, we evaluated the basal and apical secretions of TNFα, IL-6, and IL-1ß in primary RPE cells and RPE/choroid explant cells under basolateral stimulation of TLR2, 3, and 4; the effects on barrier function; and their influence on neuronal cell viability. METHODS: RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers; primary porcine RPE cells on transwell plates. Cells were basally stimulated with agonists Pam2CSK4 (Pam; TLR2), polyinosinic/polycytidylic acid (Poly I:C; TLR3), and lipopolysaccharide (LPS; TLR4) for 24 h. Supernatants were evaluated with ELISA for cytokines TNFα, IL-6, and IL-1ß. Apical supernatants were applied to SHSY-5Y cells, and cell viability was evaluated in MTT assay. Barrier function was tested by measuring transepithelial electrical resistance (TER) and occludin immunostaining. RESULTS: None of the tested TLR agonists was toxic on RPE cells after 24 h of exposure. Unstimulated RPE cells secreted hardly any cytokines. Pam induced IL-6, IL-1ß, and TNFα on the basal and apical sides at all concentrations tested. Poly I:C induced IL-6 and TNFα primarily at the basal side at lower but on both sides at higher concentrations. LPS induced IL-6, IL-1ß, and TNFα apically and basally at all concentrations tested. In the RPE/choroid, a strong difference between apical and basal secretions could be found. IL-6 was constitutively secreted basally, but not apically, but was induced by all agonists on both sides. IL-1ß and TNFα alpha were strongly induced on the basal side by all agonists. TER was reduced by all agonists, with Pam and LPS being effective in all concentrations tested. Occludin expression was unaltered, but the distribution was influenced by the agonists, with a less distinct localization at the cell borders after treatment. None of the agonists or supernatants of treated RPE and RPE/choroid organ cultures exerted any effect on viability of SHSY-5Y cells. CONCLUSIONS: Danger signals activating TLRs can induce polarized cytokine expression and contribute to the loss of barrier function in the RPE.

Self-examination low-cost full-field OCT (SELFF-OCT) for patients with various macular diseases.

PURPOSE: The treatment guidelines for many macular diseases rely on frequent monitoring with optical coherence tomography (OCT). However, the burden of frequent disease control leads to low therapy adherence in real life. OCT home monitoring would address this issue but requires an inexpensive and self-operable device. With self-examination low-cost full-field OCT (SELFF-OCT), our group has introduced a novel technology that may fulfill both requirements. In this pilot study, we report the initial experiences with a clinical prototype. METHODS: Fifty-one patients with different macular diseases were recruited in a cross-sectional study. The most common diseases were age-related macular degeneration (AMD; 39/51), diabetic macular edema (DME; 6/51), and retinal vein occlusion (RVO; 3/51). Patients received a short training in device usage and then performed multiple self-scans with the SELFF-OCT device. For comparison, scans with a standard clinical spectral domain (SD-)OCT were taken. RESULTS: After a brief training, 77% of the patients were able to successfully acquire images that were clinically gradable. No significant influence on success could be found for age (p = 0.08) or BCVA (p = 0.97). Relevant disease biomarkers in the most common retinal diseases could be detected. CONCLUSIONS: SELFF-OCT was used successfully for retinal self-examination and in the future could be used for retinal home monitoring. Future improvements in technology are expected to improve success rates and image quality. TRIAL REGISTRATION: The Trial was registered in the German Trial Register under the number DRKS00013755 on 14.03.2018.

Patients' perspective on emergency treatment of ophthalmologic diseases during the first phase of SARS-CoV2 pandemic in a tertiary referral center in Germany - the COVID-DETOUR questionnaire study.

BACKGROUND: During the first wave of the COVID-19 pandemic, the need of treatment of urgent ophthalmological diseases and the possible risk of a SARS-CoV-2 infection had to be weighed against each other. In this questionnaire study, we aimed to analyze potential barriers and patients' health beliefs during and after the lockdown early 2020 in a tertiary referral center in Kiel, Germany. METHODS: Patients admitted for the treatment of urgent ophthalmic diseases between March 1st, 2020, and June 3rd, 2020, were asked to participate in a questionnaire study. After informed consent was obtained, patients were interviewed using a standardized questionnaire which addressed aspects of their medical history, their health beliefs concerning the COVID-19 pandemic and barriers on their way to the treatment center. The study group was subdivided into two subgroups, depending on the occurrence of their symptoms, before and after the lockdown was ended on April 20th, 2020. RESULTS: Ninety-three patients were included, 43 in subgroup A (before April 20th) and 50 in subgroup B (April 20th or later). Retinal disorders were the most common causes for admission (approximately 60%).. Only 8 patients (8.6%) experienced a delay between their decision to visit a doctor until the actual examination. Every fourth patient was afraid of a COVID-19 infection, and expected a higher likelihood for an infection at the hospital. Patients with comorbidities tended to be more likely to be afraid of an infection (correlation coefficient 0.183, p = 0.0785) and were significantly more likely to be concerned about problems with organizing follow-up care (corr. Coefficient 0.222, p = 0.0328). Higher age was negatively correlated with fear of infection (corr. Coefficient - 0.218, p-value 0.034). CONCLUSION: In this questionnaire study, only a minority of patients indicated a delay in treatment, regardless of whether symptoms occurred before or after the lockdown before April 20th, 2020. While patients with comorbidities were more concerned about infection and problems during follow-up care, patients of higher age - who have a higher mortality - were less afraid. Protection of high-risk groups should be prioritized during the SARS-CoV-2 pandemic. TRIAL REGISTRATION: The study was registered as DRKS00021630 at the DRKS (Deutsches Register Klinischer Studien) before the conduction of the study on May 5th, 2020.

Selective retina therapy and thermal stimulation of the retina: different regenerative properties - implications for AMD therapy.

BACKGROUND: Selective Retina Therapy (SRT), a photodisruptive micropulsed laser modality that selectively destroys RPE cells followed by regeneration, and Thermal Stimulation of the Retina (TSR), a stimulative photothermal continuous wave laser modality that leads to an instant sublethal temperature increase in RPE cells, have shown therapeutic effects on Age-related Macular Degeneration (AMD) in mice. We investigate the differences between both laser modalities concerning RPE regeneration. METHODS: For PCR array, 6 eyes of murine AMD models, apolipoprotein E and nuclear factor erythroid-derived 2- like 2 knock out mice respectively, were treated by neuroretina-sparing TSR or SRT. Untreated litter mates were controls. Eyes were enucleated either 1 or 7 days after laser treatment. For morphological analysis, porcine RPE/choroid organ cultures underwent the same laser treatment and were examined by calcein vitality staining 1 h and 1, 3 or 5 days after irradiation. RESULTS: TSR did not induce the expression of cell-mediators connected to cell death. SRT induced necrosis associated cytokines as well as inflammation 1 but not 7 days after treatment. Morphologically, 1 h after TSR, there was no cell damage. One and 3 days after TSR, dense chromatin and cell destruction of single cells was seen. Five days after TSR, there were signs of migration and proliferation. In contrast, 1 h after SRT a defined necrotic area within the laser spot was seen. This lesion was closed over days by migration and proliferation of adjacent cells. CONCLUSIONS: SRT induces RPE cell death, followed by regeneration within a few days. It is accompanied by necrosis induced inflammation, RPE proliferation and migration. TSR does not induce immediate RPE cell death; however, migration and mitosis can be seen a few days after laser irradiation, not accompanied by necrosis-associated inflammation. Both might be a therapeutic option for the treatment of AMD.

Response of Retinal Pigment Epithelium (RPE)-Choroid Explants to Thermal Stimulation Therapy of the RPE (TSR).

BACKGROUND AND OBJECTIVES: The thermal stimulation therapy of the retinal pigment epithelium (TSR) is a sublethal laser technique for thermal stimulation of the retinal pigment epithelium (RPE)-Bruch's membrane (BrM)-complex. The aim of this study was to investigate the influence of TSR on the release of age-related macular degeneration (AMD)-relevant cell mediators. STUDY DESIGN/MATERIALS AND METHODS: Porcine RPE-BrM-choroid explants were irradiated with a 532 nm continuous wave laser using different spot sizes (100-300 µm, duration 100 milliseconds, 15-100 mW). Cell death was investigated by calcein staining. Explants were treated with grids of sublethal spots and cultivated in modified Ussing chambers. The effect on matrix metalloproteinase-2 (MMP-2) and -9 was investigated by zymography and quantitative reverse transcription polymerase chain reaction. Secretion of vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF), and transforming growth factor-ß (TGF-ß) was analyzed by enzyme-linked immunosorbent assay and expression of HSP70 was examined by western blot. Integrity of the RPE/BrM-complex was analyzed by scanning electron microscopy. RESULTS: Laser powers of 15 mW (100 µm) and 45 mW (300 µm) did not induce RPE cell death. The integrity of the RPE/BrM-complex was not impaired after TSR. After TSR with 300 µm spot size, we observed a significant increase of active MMP-2 in the basal compartments. The content of PEDF significantly increased in treated explants in both compartments with 100 and 300 µm spot sizes. VEGF and TGF-ß secretion was not triggered by TSR. CONCLUSIONS: TSR represents a possible RPE stimulating treatment for dry AMD. TSR increases the basal release of active MMP-2, which might reverse age-related thickening of BrM. VEGF secretion was not triggered by TSR while anti-angiogenic PEDF was increased, indicating an induction of an anti-angiogenic and neuroprotective environment. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Differences in Uptake and Intracellular Fate between Bevacizumab and Aflibercept after Repetitive Long-Term Treatment in the Retinal Pigment Epithelium.

INTRODUCTION: Anti-VEGF therapy is repeatedly given for an extended period of time to patients when treated for age-related macular degeneration. While short-term effects of anti-VEGF agents on retinal pigment epithelial cells have been investigated, the effects of long-term and repeated treatment on these cells are scarce. In this study, we have investigated the effects of anti-VEGF treatment (bevacizumab and aflibercept) after long-term, repeated treatment on uptake, storage, and subcellular localization. METHODS: Experiments were conducted in primary porcine retinal pigment epithelium (RPE) cells in first passage and in ARPE-19 cell line. Cells were treated with 250 µg/mL bevacizumab, aflibercept, or, as a non-VEGF inhibiting antibody, rituximab once a week for 1 day, 7 days, 4, and 12 weeks. Cell survival was evaluated with methyl thiazolyl tetrazolium assay. Uptake and localization of compounds were investigated with immunofluorescence microscopy. Selective intracellular proteins were stained with specific respective primary antibodies; actin cytoskeleton was stained with phalloidin. For quantitative analysis, intracellular signals were normalized to light intensity and exposure time. Intracellular association with lysosomes (Lamp2) and exosomes (CD63) was also quantified. In addition, subcellular fractions (nucleus, plasma, membrane, and cytoskeleton) were generated and analyzed in Western blot. RESULTS: Weekly treatment up to 12 weeks displayed no toxic effects on RPE cells in any substance tested. Intracellular signal of bevacizumab and aflibercept was strongest after 1 day, decreased after 1 and 4 weeks but increased again after 12 weeks. The signal of intracellular bevacizumab was significantly stronger than of aflibercept. In addition, in primary RPE, aflibercept was significantly more associated with Lamp2, indicating degradation of aflibercept. At all time points, the respective therapeutics could be detected at the cytoskeleton. In primary RPE cells, co-localization with exosome marker CD63 showed a maximum after 1 day for bevacizumab and after 12 weeks for aflibercept. Actin-encapsulated therapeutics can be found at any time point tested. CONCLUSION: Both bevacizumab and aflibercept display a distinctive time-dependent uptake in the RPE cells and are stored in actin-covered accumulations for extended periods of time. When normalized and quantified, less aflibercept can be found in RPE cells, while more aflibercept is co-localized with Lamp2. Our data suggest that bevacizumab is differently processed by RPE cells than aflibercept.

Evaluation of the Effects of Fucoidans from Fucus Species and Laminaria hyperborea against Oxidative Stress and Iron-Dependent Cell Death.

Fucoidans are algal polysaccharides that exhibit protective properties against oxidative stress. The aim of this study was to investigate different fucoidans from brown seaweeds for their ability to protect against iron-dependent oxidative stress (ferroptosis), a main hallmark of retinal and brain diseases, including hemorrhage. We investigated five new high-molecular weight fucoidan extracts from Fucus vesiculosus, F. serratus, and F. distichus subsp. evanescens, a previously published Laminaria hyperborean extract, and commercially available extracts from F. vesiculosus and Undaria pinnatifida. We induced oxidative stress by glutathione depletion (erastin) and H2O2 in four retinal and neuronal cell lines as well as primary cortical neurons. Only extracts from F. serratus, F. distichus subsp. evanescens, and Laminaria hyperborea were partially protective against erastin-induced cell death in ARPE-19 and OMM-1 cells, while none of the extracts showed beneficial effects in neuronal cells. Protective fucoidans also attenuated the decrease in protein levels of the antioxidant enzyme GPX4, a key regulator of ferroptosis. This comprehensive analysis demonstrates that the antioxidant abilities of fucoidans may be cell type-specific, besides depending on the algal species and extraction method. Future studies are needed to further characterize the health-benefiting effects of fucoidans and to determine the exact mechanism underlying their antioxidative abilities.

Retinal Pigment Epithelium Expressed Toll-like Receptors and Their Potential Role in Age-Related Macular Degeneration.

(1) Background: Inflammation is a major pathomechanism in the development and progression of age-related macular degeneration (AMD). The retinal pigment epithelium (RPE) may contribute to retinal inflammation via activation of its Toll-like receptors (TLR). TLR are pattern recognition receptors that detect the pathogen- or danger-associated molecular pattern. The involvement of TLR activation in AMD is so far not understood. (2) Methods: We performed a systematic literature research, consulting the National Library of Medicine (PubMed). (3) Results: We identified 106 studies, of which 54 were included in this review. Based on these studies, the current status of TLR in AMD, the effects of TLR in RPE activation and of the interaction of TLR activated RPE with monocytic cells are given, and the potential of TLR activation in RPE as part of the AMD development is discussed. (4) Conclusion: The activation of TLR2, -3, and -4 induces a profound pro-inflammatory response in the RPE that may contribute to (long-term) inflammation by induction of pro-inflammatory cytokines, reducing RPE function and causing RPE cell degeneration, thereby potentially constantly providing new TLR ligands, which could perpetuate and, in the long run, exacerbate the inflammatory response, which may contribute to AMD development. Furthermore, the combined activation of RPE and microglia may exacerbate neurotoxic effects.

Effect of long-term inflammation on viability and function of RPE cells.

PURPOSE: Degenerative ocular disorders like age-related macular degeneration (AMD) are associated with long-term pro-inflammatory signals on retinal pigment epithelial (RPE) cells. In this study, we investigated the effect of long term treatment of RPE cells with agonists of toll-like receptor (TLR) -3 (Polyinosinic:polycytidylic acid, Poly I:C), TLR-4 (lipopolysaccharide, LPS) and the pro-inflammatory cytokine TNFα. METHODS: All tests were conducted with primary porcine RPE. Cells were stimulated with Poly I:C (1, 10, 100 µg/ml), LPS (0.1, 1, 10 µg/ml) or TNFα (12.5, 25 or 50 ng/ml) for 1 day, 7 days or 4 weeks. Cell viability tests (MTT) were additionally tested in ARPE-19 cells. Cytokine secretion (IL-6, IL-1ß, IL-8, TNFα, TGF-ß) was tested in ELISA, phagocytosis in a microscopic assay, and expression of RPE65 in Western blot. Barrier function was tested in transwell-cultured cells by measuring transepithelial resistance for up to 3 days. RESULTS: LPS and TNFα significantly reduce cell viability after 1 day and 7 days, Poly I:C after 7 days and 4 weeks. LPS, Poly I:C and TNFα significantly induce the secretion of IL-6 and IL-8 at all tested time points. IL-1ß is increased by LPS and Poly I:C after 1 day, but not by TNFα. TNFα secretion is increased by Poly I:C and LPS after 1 day but not at later time points. TGF-ß secretion is not influenced by any stimulus. Concerning RPE function, LPS decreased phagocytosis after 7 days, while Poly I:C and TNFα showed no effect. RPE65 expression was strongly reduced by TNFα and LPS after 4 weeks. Wound healing capacity was reduced by Poly I:C but induced by LPS after 7 d and 4 w. Barrier function was not affected by Poly I:C or LPS, while TNFα reduced barrier function after 1 h, 4 h and 3 days. CONCLUSION: Long term pro-inflammatory stimuli reduce RPE viability, barrier properties and cellular function and induce pro-inflammatory cytokines and therefore may contribute directly to atrophic changes in AMD.