Preventive Effects of Omega-3 and Omega-6 Fatty Acids on Peroxide Mediated Oxidative Stress Responses in Primary Human Trabecular Meshwork Cells

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease

Assessing the adherence behavior of glaucoma patients to topical eye drops

Ulrich Welge-Lussen,1 Stefanie Weise,2 Alice L Yu3 1Department of Ophthalmology, Friedrich-Alexander University, Erlangen, Germany; 2Department of Ophthalmology, University of Cologne, Cologne, Germany; 3Department of Ophthalmology, Ludwig Maximilian University, Munich, Germany Purpose: The goal of this study was to determine the adherence of glaucoma patients to their topical glaucoma medication. Furthermore, the relationships between the adherence behavior and the patients’ demographic data, clinical characteristics, and their knowledge about glaucoma were evaluated. Methods: This was a prospective study of 123 patients with primary open-angle glaucoma who were given two standardized questionnaires. The first questionnaire at time point T1 comprised a knowledge assessment and the self-reported adherence measures Adherence to Refills and Medication Scale 2 (ARMS2), visual analogue scale for adherence (VAS-AD), and missed doses in the past 14 days. Two months later at time point T2, a second questionnaire reevaluated the adherence measures ARMS2, VAS-AD, and missed doses in the past 14 days. Results: There was a good correlation among all the three adherence measures at T1 and T2. The mean values of ARMS2 were in the lower range, with 3.38 at T1 and 2.8 at T2. The VAS-AD detected that 18.5% of patients always took their eye drops correctly, and 77.9% of patients reported not to have missed a single dose in the past 14 days. There was no significant correlation between the patients’ demographic data or knowledge about glaucoma and the adherence measures ARMS2 or VAS-AD. Among the clinical characteristics, only single-eye blindness showed a significant correlation with VAS-AD. Conclusion: In this study, no general relationships were found between medication adherence and the patients’ demographic data, clinical characteristics, or knowledge about glaucoma. It may be assumed that more individualized strategies are required to optimize adherence behavior. Keywords: adherence, eye drops, glaucoma, medicatio

Intensity of side effects of topical glaucoma medication and its influence on adherence behavior in patients with glaucoma

Alice L Yu,1 Stefanie Weise,2 Ulrich Welge-Lussen3 1Department of Ophthalmology, Ludwig-Maximilians-University, Munich, 2Department of Ophthalmology, University of Cologne, Cologne, 3Department of Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany Background: The aim of this study was to investigate the intensity of side effects that patients attribute to their topical glaucoma medication and their relationship to adherence behavior. Methods: This was a questionnaire-based study of 123 glaucoma patients at a university eye clinic in Erlangen, Germany. An initial questionnaire asked about patient demographic data, the treatment plan, and intensity of side effects, and included Adherence to Refills and Medication Scale 2 (ARMS2) and visual analog scale (VAS-AD) scores. In a follow-up questionnaire, the treatment plan, intensity of side effects, ARMS2, and VAS-AD were reanalyzed. Results: Most patients reported having few side effects, although only 20% said that they had no symptoms suggestive of side effects at all. The patients showed good adherence behavior on both the ARMS2 and VAS-AD scores, which were stable over time. The intensity of side effects experienced in the previous 7 days did not correlate with adherence scores and had no predictive value for adherence. Conclusion: This study could not detect any significant influence of the subjectively experienced intensity of side effects on patients’ adherence behavior. However, we believe that a simple and clear treatment plan with few side effects is still preferred by most patients. Keywords: adherence, side effects, eye drops, glaucom

Factors affecting the use of antioxidant supplements in patients with late AMD

Alice L Yu,1 Tobias Paul,1 Markus Schaumberger,1 Ulrich Welge-Lussen2 1Department of Ophthalmology, Ludwig-Maximilians-University, Munich, 2Department of Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany Background: The purpose of this study was to assess the use of oral antioxidant supplements in patients with late age-related macular degeneration (AMD) and to identify influencing factors that may affect the use of such supplements. Methods: The study included 47 patients with late AMD. Using a questionnaire, the patients were asked for their demographic, ophthalmologic, and systemic data, their source of recommendation of antioxidant use for AMD, and/or their reasons for nonuse. The demographic, ophthalmologic, and systemic information was correlated with use or nonuse of oral antioxidant supplements for AMD. Results: Sixty-eight percent (32/47) of patients took antioxidant supplements for AMD and 32% (15/47) of patients did not. There were no statistically significant differences in demographic, ophthalmologic, and systemic parameters between patients with late AMD who used supplements and those who did not. Two thirds of patients with late AMD (66%, 31/47) reported being recommended oral antioxidant supplements for AMD by their ophthalmologist. Patients who did not use antioxidant supplements either did not obtain any recommendation or did not believe in their benefits. Conclusion: This study shows that most patients with late AMD use antioxidant supplements despite the recommendation to do so being missing in the Age-Related Eye Disease Study. Our study emphasizes the importance of seeking further therapeutic options for patients with late AMD. Keywords: age-related macular degeneration, antioxidants, micronutrients, Age-Related Eye Disease Stud

Evaluation of intraocular pressure elevation after multiple injections of intravitreal ranibizumab

Alice L Yu,1,* Florian Seidensticker,1,* Markus Schaumberger,1 Ulrich Welge-Lussen,2 Armin Wolf11Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; 2Department of Ophthalmology, Friedrich-Alexander-University, Erlangen, Germany*These authors contributed equally to this workBackground: We wanted to determine whether multiple injections of intravitreal ranibizumab was associated with an elevated intraocular pressure (IOP) in patients treated for neovascular age-related macular degeneration (AMD).Methods: This retrospective study examined 53 patients with neovascular AMD treated with multiple injections of intravitreal ranibizumab. The main outcome measure was the difference in IOP between the frequently-treated study eyes (≥15 injections) and the unfrequently-treated fellow control eyes (≤ five injections). Patients were divided into three study groups: group I (35 patients with 15 to 19 injections); group II (15 patients with 20 to 29 injections); and group III (three patients with ≥30 injections). The IOP was measured by Goldmann applanation tonometry 4 weeks after the last injection of intravitreal ranibizumab. For statistical analysis, the IOP was then correlated with the number of ranibizumab injections.Results: Among the frequently-treated study eyes, the mean IOP was 13.68±2.91 mmHg (range, 8 to 20 mmHg). The unfrequently-treated fellow control eyes had a mean IOP of 13.45±3.09 mmHg (range, 9 to 25 mmHg). There was no significant correlation of the IOP difference between the study and control eyes with the number of ranibizumab injections (correlation coefficient 0.77; P=0.583). For each of groups I, II, and III, the difference in mean IOP between the study and control eyes was nonsignificant (P>0.05). There was also no significant association of the IOP difference between the study and control eyes with the number of ranibizumab injections for each group (P=0.391).Conclusion: Our study did not find an increased IOP in frequently-ranibizumab-treated eyes when compared to unfrequently-treated fellow control eyes. Further studies with a greater sample size are needed to evaluate whether an increased number of ranibizumab injections is associated with IOP changes.Keywords: age-related macular degeneration, ocular hypertension, anti-VEGF therap

Ultrastructure of the vitreoretinal interface following plasmin assisted vitrectomy

AIMS—To investigate the ultrastructure of the vitreoretinal interface following plasmin induced posterior vitreous detachment.
METHODS—Plasmin (1 or 2 U/0.1 ml) was injected into the vitreous cavity of 24 eyes of freshly slaughtered pigs. The 24 fellow eyes received calcium-free and magnesium-free PBS and served as a control. After incubation at 37°C for 30 and 60 minutes, the globes were placed in fixative and hemisected. Specimens for light, scanning, and transmission electron microscopy were obtained from the posterior pole, the equator, and the vitreous base using a corneal trephine.
RESULTS—All plasmin treated eyes showed posterior vitreous detachment. However, the inner limiting membrane (ILM) was covered by remnants of cortical vitreous at the posterior pole and at the equator. There was a direct correlation between the concentration and exposure times of plasmin and the degree of vitreoretinal separation. Eyes exposed to 1 U plasmin for 30 minutes had a dense network of residual collagen fibrils while those exposed to 1 U plasmin for 60 minutes had only sparse collagen fibrils covering the ILM. Eyes treated with 2 U plasmin for 60 minutes had a smooth retinal surface, consistent with a bare ILM. At the vitreous base there was no vitreoretinal separation. In all control eyes the vitreous cortex was completely attached to the retina. There was no evidence of retinal damage in any plasmin treated eye.
CONCLUSION—Plasmin induces a cleavage between the vitreous cortex and the ILM without morphological changes to the retina. In contrast with previous reports, plasmin produces a smooth retinal surface and additional surgery is not required in this experimental setting. The degree of vitreoretinal separation depends on the concentration and length of exposure to plasmin.


Macular pigment optical density measured by heterochromatic modulation photometry

PURPOSE:To psychophysically determine macular pigment optical density (MPOD) employing the heterochromatic modulation photometry (HMP) paradigm by estimating 460 nm absorption at central and peripheral retinal locations. METHODS:For the HMP measurements, two lights (B: 460 nm and R: 660 nm) were presented in a test field and were modulated in counterphase at medium or high frequencies. The contrasts of the two lights were varied in tandem to determine flicker detection thresholds. Detection thresholds were measured for different R:B modulation ratios. The modulation ratio with minimal sensitivity (maximal threshold) is the point of equiluminance. Measurements were performed in 25 normal subjects (11 male, 14 female; age: 30 ± 11 years, mean ± sd) using an eight channel LED stimulator with Maxwellian view optics. The results were compared with those from two published techniques - one based on heterochromatic flicker photometry (Macular Densitometer) and the other on fundus reflectometry (MPR). RESULTS:We were able to estimate MPOD with HMP using a modified theoretical model that was fitted to the HMP data. The resultant MPODHMP values correlated significantly with the MPODMPR values and with the MPODHFP values obtained at 0.25° and 0.5° retinal eccentricity. CONCLUSIONS:HMP is a flicker-based method with measurements taken at a constant mean chromaticity and luminance. The data can be well fit by a model that allows all data points to contribute to the photometric equality estimate. Therefore, we think that HMP may be a useful method for MPOD measurements, in basic and clinical vision experiments

Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells.

PURPOSE: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. METHODS: Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. RESULTS: One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. CONCLUSIONS: Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation