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Clinical ophthalmology Volume 11, 621-629 (2017). doi:10.2147/OPTH.S123513, Published by Dove Medical Press, Auckland, New Zealand

Ayşegül Tura,1 Aizhan Alt,1 Julia Lüke,1 Salvatore Grisanti,1 Christos Haritoglou,2 Carsten H Meyer,3 Khaled Nassar,1 Matthias Lüke1 On behalf of the International Chromovitrectomy Collaboration 1Department of Ophthalmology, University of Schleswig-Holstein, Lübeck, Germany; 2Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany; 3Department of Ophthalmology, PallasClinic, Aarau, Switzerland Purpose: To examine the viability and differentiation of retinal pigment epithelial (RPE) cells after exposure to the vital dye Acid Violet-17 (AV-17). Methods: Bovine RPE cells were incubated with AV-17 (0.0625–0.5 mg/mL) for 30 seconds or 5 minutes. Viability was determined by live/dead staining, cleaved CASP3 immunostainings, and MTT test. Actin cytoskeleton was visualized by Alexa 488-phalloidin. Immunocytochemistry was performed to determine the levels of ZO-1, CTNNB1, and KRT19. Results: Exposure to AV-17 at the concentrations of 0.25–0.5 mg/mL resulted in a dose-dependent decrease in viability, the loss of ZO-1 from tight junctions, translocation of CTNNB1 into the cytoplasm and nucleus, disarrangement of the actin cytoskeleton, and a slight increase in KRT19. Conclusion: AV-17 at a concentration

BACKGROUND To evaluate the regulation of blood supply in primary uveal melanomas through caveolin-1 (Cav-1)/phosphoinositol-3 kinase (PI3K). METHODS The expression of Cav-1 and PI3K was analysed in 51 paraffin sections of metastatic (n = 30) and non-metastastic uveal melanomas (n = 21). Two trained observers quantified Cav-1 and PI3K immunofluorescensce expression by determining intensity of staining and percentage of positive cells. The expression was correlated with known prognostic factors. Besides angiogenesis by means of endoglin expression, the normal vasculature (von Willebrand Factor expression) was evaluated semi-quantitatively. Vasculogenic mimicry (VM) was analysed by CD31/PAS staining. RESULTS All examined specimens expressed Cav-1 with a mean of 90.34% Cav-1 positive cells (range, 3.23-100%). Metastatic disease was associated with a higher Cav-1 expression. The correlation of Cav-1 with well-established prognostic factors showed a significant association between Cav-1 expression and largest tumour diameter (P = 0.022), tumour node metastasis classification (P = 0.008) and invasion of optic nerve head (P = 0.048). PI3K was expressed by all uveal melanomas with a mean of 87.28% cells showing PI3K expression. A higher level of PI3K was significantly associated with larger height (P = 0.042) and progressed tumour node metastasis stage (P = 0.016). The percentage of PI3K and Cav-1 positive cells were significantly associated (P = 0.034). For PI3K and Cav-1 expression a non-significant association with VM was shown (P = 0.064 and P = 0.072, respectively). No correlation of PI3K or Cav-1 with angiogenesis or mature vasculature was seen (P > 0.05). CONCLUSIONS Cav-1 expression may be especially up-regulated in larger uveal melanomas. As it was correlated with PI3K expression and VM in this series of uveal melanoma, Cav-1 might induce the formation of VM via the PI3K-signalling cascade.

Hintergrund: Bisher ist die genaue Expression des Ephrin-Typ-A2-Rezeptors (EphA2) im Hinblick auf dessen Bedeutung wahrend der Zell-Zell-Adhasion, Zellmigration, Angiogenese und der Formation von VM-Kanalen (VM: Vasculogenic Mimicry) in uvealen Melanomen unter Berucksichtigung der Metastasierungsrate unklar. Material und Methoden: Paraffinschnitte von 50 histopathologisch gut charakterisierten Fallen primarer uvealer Melanome (durchschnittlicher Tumordurchmesser: 16,3 mm) wurden hinsichtlich ihrer Expression von EphA2 evaluiert. Bei 29 Patienten traten systemische Metastasen auf. Die verbleibenden 21 Patienten hatten einen durchschnittlichen Nachuntersuchungszeitraum von 10 Jahren. Zusatzlich wurde die Tumorangiogenese mittels Endoglin-Expression (CD105), das mature Gefassystem (Von-Willebrand-Faktor, vWF) und das Vorhandensein von VM (CD31/PAS-Farbung) untersucht. Ergebnisse: Alle uvealen Melanome exprimierten EphA2 mit einer durchschnittlichen Anzahl positiver Zellen von 95,93 % (± Standardabweichung: 6,3 %). Es war zwar keine signifikante objektive Assoziation von EphA2 und der Metastasierungsrate (p = 0,196) bzw. der Endoglin-Expression (p = 0,652), VM (p = 0,267) oder weiteren klinischen oder histopathologischen Prognosefaktoren (p

To determine the short-term safety of human recombinant decorin protein in preventing proliferative vitreoretinopathy (PVR) in perforating injuries. This is a prospective, single-center, open-label, interventional case series. Single intravitreal injection of decorin 200 μg (n = 4) or 400 μg (n = 8) was given 48 h after injury. At the tenth day, pars plana vitrectomy was done whenever indicated. Flash electroretinogram (ERG) was done before and 3 months post-injection. We assessed ocular inflammation, ERG changes, and retinal layer integrity by optical coherence tomography (OCT). Systemic and vitreous pharmacokinetics were also evaluated. Twelve patients (12 eyes) with perforating globe injuries (zone III) were included and followed for a median of 6 months. Intravitreal decorin injection was well tolerated with no ocular or systemic safety adverse events. Decorin retinal safety was demonstrated anatomically by intact retinal layer by OCT, and functionally by flash ERG which did not show any significant worsening during the study and the final mean logMAR best-corrected visual acuity (BCVA) which was 1.15 (20/280) and 0.7 (20/100) for groups A and B, respectively, and ≥ 20/200 in 75% of all eyes. Decorin serum and vitreous levels were elevated following trauma, with higher and extended levels following intravitreal injection. No short-term safety concerns were detected after a single intravitreal injection of decorin in patients with perforating injuries. ClinicalTrials.gov ID: NCT02865031

Background: Dye solutions such as indocyanine green (ICG) are used for the staining of intraocular structures. The aim of the presented study was to investigate the effects of ICG on bovine retinal function using different concentrations of ICG. Methods: Bovine retina preparations were perfused with a standard solution and the electroretinogram was recorded. The nutrient solution was substituted by an ICG solution at varying concentrations for 45 min. Afterwards the preparations were reperfused with standard solution for at least 85 min. Results: Significant reductions in b-wave amplitude were found for concentrations of 0.0025% (p = 0.0099) and 0.025% (p = 0.0378). For the concentration of 0.025%, the b-wave amplitude remained significantly decreased (p = 0.0082) after the observation period, but a full recovery of the b-wave was observed for the concentration of 0.0025% (p = 0.1917). Conclusion: Intraocular application of sufficient ICG concentrations for internal limiting membrane staining seems not possible without interfering with retinal function.

To evaluate the capability of adjuvant intraocular ranibizumab (Lucentis®) injections in the treatment of rubeosis and intraocular pressure in patients with rubeosis and neovascular glaucoma. Ten eyes with rubeosis (R) and ten eyes with neovascular glaucoma (NVG) received Lucentis® injections (ranibizumab 0.5 mg/0.05 ml) in this prospective, monocenter, 12-months, interventional case series. The primary efficacy outcome measure was the change of degree of iris rubeosis as documented by iris fluorescein angiography measured after 12 months. Secondary outcomes were intraocular pressure (IOP), best-corrected visual acuity (BCVA, logMAR), numbers of additional interventions or antiglaucoma medications administered after injection, the gonioscopic status of the anterior chamber angle, and central retinal thickness. In the R group, 3.6 injections and in the NVG group 2.3 injections of Lucentis® were administered. Additional treatments were photocoagulation (n = 19), cyclodestructive procedures (n = 9), cryopexy (n = 3), and vitrectomy (n = 1). The mean stage of rubeosis was 3.4 ± 0.7 in the R group and 3.6 ± 0.8 in the NVG group at baseline. At month 12, the rubeosis was almost resolved in the R group (0.1 ± 0.3, p

The aim of the study was to analyze the effect of Dimethylenaston, a mitotic kinesin 5 (Eg5) inhibitor, in an experimental setting of glaucoma filtration surgery.On 37 chinchilla rabbits (ChBBCH), glaucoma filtration surgery similar to clinical practice, was performed. The animals received either no adjuvant, one unilateral subconjunctival injection of Dimethylenastron (1.0 µmol, 3.0 µmol), or the vehicle alone at baseline and in two further groups additionally at days 3 and 7 thereafter (1.0 µmol, 3.0 µmol). The evaluation of antifibrotic efficacy was performed by clinical response, histological examination, and immunohistochemical staining for smooth muscle actin (SMA) and CD 31. The animals were sacrificed on day 14, and the eyes processed for histology.The vehicle was well tolerated. Except for two cases of transient fibrinous reaction after the injection of 3.0 µmol Dimethylenastron, no adverse effects, such as inflammation or blurring of the optical media, were observed. A bleb scarring occurred in the group that received surgery only, adjuvant DMSO, or Dimethylenastron 3.0 µmol. Dimethylenastron (1.0 µmol) induced a milder scarring compared with the control group but the length of bleb survival was not significantly prolonged (p = 0.053, Kaplan-Meier log rank test). In all groups, the intraocular pressure correlated with the fibrotic process and reached normal levels within 14 days after surgery. Those groups injected with 1.0 µmol Dimethylenastron revealed a significantly reduced ratio of intraocular pressure and a milder, but not sufficiently reduced, subconjunctival fibrotic reaction according to the histological and immunohistochemical analysis.The subconjunctival administration of Dimethylenastron 1.0 µmol induced a milder conjunctival scarring. The applied concentrations of Dimethylenastron did not improve the surgical outcome of glaucoma filtration treatments in rabbits sufficiently.

To evaluate the short-term toxic effects of pegaptanib sodium on retinal function. At present, intraocular anti-vascular endothelial growth factor (VEGF) therapy is the primary choice of treatment for neovascular maculopathy. The isoform VEGF(165) is specifically inhibited by pegaptanib sodium. Therefore, since VEGF(165) has neuroprotective effects against apoptosis of neuronal cells, blockage of VEGF(165) by pegaptanib could induce retinal dysfunction. In the present study, we used an electrophysiological technique for testing retinal toxicity in order to evaluate the short-term toxic effects of pegaptanib sodium on retinal function in a model of isolated perfused vertebrate retina.Isolated bovine retinas were perfused with an oxygenated, pre-incubated nutrient solution. Electroretinograms (ERGs) were recorded as trans-retinal potentials using Ag/AgCl electrodes. Pegaptanib sodium (0.006, 0.06, or 0.2 mg/ml) and solvent carrier were added to the nutrient solution for 45 min. ERGs were monitored before, during, and after exposure.No significant reductions of b-wave (p = 0.357, p = 0.31, and p = 0.11, respectively) or a-wave (p = 0.189, p = 0.46, and p = 0.23, respectively) amplitudes were detected during application of pegaptanib (0.006, 0.06, or 0.2 mg/ml). The solvent carrier alone had no effect on ERG b- or a-waves (p = 0.98 and p = 0.42, respectively). During washout, ERG amplitudes of all test series remained unchanged.Results suggest that both pegaptanib sodium and its solvent carrier have good safety profiles. Intraocular application of 0.3 mg pegaptanib sodium induced no significant changes in ERGs in our ex vivo model and, thus, appears to be safe.

To evaluate the expression of melanoma cell adhesion molecule (MCAM) as prognostic factor in uveal melanoma patients.Paraffin sections from 35 primary uveal melanomas were analyzed immunhistochemically for the expression of MCAM. Further known prognostic factors such as cell type, ciliary body invasion, tumor stage, and local extension were evaluated. In 16 cases, patients had developed metastatic disease. The patients without metastasis (n = 19) had a mean follow-up of 10.6 (9-13) years.All tumors were positive for MCAM (mean: 57.05%). In the group without metastasis, an average MCAM expression of 35.37% (SD 19.66%) compared to 82.78% (SD 11.73%) in the metastasis group was found. The Cox regression analysis showed a significant association of an enhanced MCAM expression and death due to metastasis (HR: 365.48, 95% CI: 18.51, 7217.85).MCAM expression of primary uveal melanomas can serve as a possible prognostic parameter for metastasis. If further experiments approve the cellular adhesion molecules to account for early micrometastasis of tumor cells, a possible target might be available for future treatment.

Appropriate fixation of the eye is crucial for the evaluation of pathological changes such as retinal detachment (RD) in animal models. An ideal fixative should have a rapid penetration ability that can fix the delicate structures of the eye without disturbing the interfaces. Formaldehyde is a widely used, rapidly penetrating fixative. However, it takes at least a week to achieve full stabilization of histological structures. As well, whole eyes fixed in 4% buffered formaldehyde demonstrate a variety of artefacts, including separation of the neurosensory retina from the retinal pigment epithelium (RPE) (Kiernan 1999). Few chemicals have challenged the pre-eminence of 4% buffered formaldehyde as an ocular fixative. Among them is Bouin's fluid (BF), which is a mixture of different chemically active ingredients: picric acid, acetic acid and formaldehyde. However, the presence of picric acid in the fixative leads to safety hazards and disposal problems. Also, for an optimal fixation with BF, several alcohol rinses over two days are required and need to be followed by repeated incubation with an alcohol–lithium carbonate solution until the yellow colour of BF is depleted from the tissue (Cleary & Ryan 1979a,b; Latendresse et al. 2002). Another option for fixation was suggested by Yanoff and Fine: they found that a mixture of 2% buffered formaldehyde and 0.5% glutaraldehyde was superior to either fixative alone and gave acceptable results for both light and electron microscope (Yanoff & Fine 1967). Margo and Lee used enucleated eyes from dogs, rabbits and pigs that had been killed for non-ocular experiments to test the fixative effects. They recommended 1% formaldehyde plus 1.25% glutaraldehyde in phosphate buffer for immersion fixation of whole untreated eyes. They concluded that this solution causes a reduction in tissue distortion, thus preventing the separation of the neurosensory retina from the RPE (Margo & Lee 1995). In our study we wanted to find out the appropriate fixative for a standardized PVR (proliferative vitreoretinopathy) model in rabbit eyes. PVR is the most common reason for failure of retinal reattachment surgery and tractional retinal detachment after ocular trauma (Holekamp & Grand 1997; Colby 1999; Wang et al. 2007). Following penetrating trauma, PVR is estimated to occur in between 10 and 45% of eyes with a mean incidence of approximately 25% (Colby 1999; Wang et al. 2007). Therefore, PVR remains a problem despite advances in vitreoretinal surgery (Blumenkranz et al. 1982; Charteris et al. 2002; Sadaka & Giuliari 2012). For better understanding of the mechanisms and to evaluate therapeutic strategies against PVR, experimental models were developed (Agrawal et al. 2007). In the current study, we evaluate the use of 1% formaldehyde containing 1.25% glutaraldehyde compared to 4% formaldehyde as a fixative. Our goal was to improve the consistency of histopathological outcomes in a previously described rabbit model of PVR (Cleary & Ryan 1979a,b), in order to have a reliable base for further drug-testing experiments.

Purpose: The aim of this study is to explore the effects of SB 202190, a highly selective p38 MAPK inhibitor, on bleb survival following glaucoma filtering surgery. Materials and Methods: Human Tenon’s fibroblasts were treated with SB 202190 (0 to 100 μM) to determine IC50, and cell proliferation and migration. Twenty rabbits were divided into 4 groups (G1-G4): G1 animals received only a trabeculectomy. G2-G4 animals had trabeculectomy plus one of the following subconjunctival adjuvants, given intraoperatively and postoperatively: G2=sham, G3=20 μM SB 202190, and G4=50 μM SB 202190. The blebs were assessed using the Indiana Bleb Appearance Grading Scale. The intraocular pressure (IOP) was expressed as the right to left eye ratio (R/L ratio). For morphometric bleb analysis the JMicrovision software was used. Results: SB 202190 inhibits human Tenon’s fibroblasts proliferation and migration in vitro (IC50=17.2 μM). In vivo subconjunctival application of SB 202190 after glaucoma filtration surgery significantly increases bleb height, bleb extension, and bleb survival time compared with the control. In all groups, the IOP ratio correlates with the fibrotic process. G3 shows a significantly reduced IOP ratio at day 14 compared with the control. Analysis of the bleb histology shows that G3 has a significant smaller fibrosis area compared with G1 and G2. Application of the highest dose (50 μM SB 202190) is associated with hyphema in 2 of 5 animals (40%). Conclusion: Application of SB 202190 significantly improves bleb characteristics and IOP control after filtering glaucoma surgery in a rabbit model.

Purpose This study evaluates the potential of serum proinflammatory cytokines as AMD biomarkers. Methods Serum samples from 30 age-related macular degeneration (AMD) patients and 15 age-matched controls were examined for 16 inflammatory cytokines using multiplex ELISA. Patients were divided into three subgroups (improvement/nochange/deteriorationduringanti-VEGFtreatment)by OCT and funduscopy, and correlated to the cytokine levels. Results Serum concentrations of IL-1α,IL-1β,IL-4,IL-5,IL10, IL-13, and IL-17 were significantly higher in AMD patients than in controls. None of the co-variables expressed a significant effect on the tested cytokines. Only IL-1a and IL17 showed a statistically significant difference between groups (improved, unchanged, deteriorated) as determined by one-way ANOVA. Patients with increased macular thickness during treatment showed significantly lower levels of IL17 compared to improved cases and to unchanged cases (p= 0.004, 0.03 respectively, Dunnett’s T3 post hoc multiple test). TNF-α was significantly higher in improved cases compared to deteriorated cases (p=0.03, Dunnett’s T3 post hoc multiple test). IL-17 was a significant predictor for macular oedema using linear regression (β=�0.888, p

This study evaluates the use of the TGF-β receptor 1 inhibitor LY-364947 (LY) to prevent proliferative vitreoretinopathy (PVR). For the in vitro experiments Human Tenon's Fibroblasts (HTFs) and retinal pigment epithelial (RPE) cells were treated with different concentrations of LY to determine HTF proliferation and RPE transdifferentiation. For in vivo testing 30 rabbits underwent a PVR trauma model. The animals received different concentrations of intravitreally injected LY, with or without vitrectomy. LY treatment reduced HTF proliferation and RPE transdifferentiation in vitro. In vivo intravitreal injection of LY prevented PVR development significantly. This positive effect was also present when LY injection was combined with vitrectomy. Intravitreal injection of LY prevented tractional retinal detachment in 14 out of 15 animals. In conclusion, treatment with the TGF-β receptor 1 inhibitor LY reduces HTF proliferation and RPE transdifferentiation in vitro and prevents proliferative vitreoretinopathy and subsequent tractional retinal detachment in vivo.

Purpose of the Study: To investigate the outcome of brilliant blue G (BBG)-assisted internal limiting membrane peeling in macular hole surgery after a follow-up of 6 months. Procedures: In this retrospective study 20 eyes have been treated with BBG-assisted macular hole surgery. After a follow-up of 6 months, the functional and anatomical outcomes have been analyzed. Results: The mean preoperative best-corrected visual acuity (BCVA) was 0.7 logMAR units (mean ± SD 0.66 ± 0.27). After 3 months, the mean BCVA increased not significantly to 0.4 (0.54 ± 0.30), but a significant improvement to 0.2 logMAR units (0.28 ± 0.23) could be detected after 6 months compared to baseline (p < 0.01). Primary macular hole closure after a single surgery was found in 17 of 20 eyes. Conclusion and Message: BBG exhibits sufficient staining qualities and safety profile leading to a significant functional improvement after successful macular hole surgery.

The cytokine transforming growth factor-s (TGF-s) is a pivotal contributor to tissue fibrosis and a key cytokine in the pathogenesis of cellular transdifferentiation, epithelial-mesenchymal transition (EMT), and cell adhesion. This study evaluates the effect of decorin, a naturally occurring TGF-s inhibitor, in an experimental rabbit model for proliferative vitreoretinopathy (PVR). Traumatic PVR was induced in 50 rabbits divided into ten groups (n = 5). One group (GI) reveals a control with no treatment after trauma. Groups (GII–GIV) consisted of subgroups receiving phacovitrectomy at three different time points; (a) at the time of trauma, (b) 1 week following trauma, and (c) 2 weeks following trauma. GIII and GIV received 100 μg or 200 μg decorin, respectively. PVR severity was scored from 0 to 4. The amount of fibrosis was quantified using JMicroVision© software. The control group GI developed severe PVR with tractional retinal detachment (TRD); (PVR score ≥2) in four rabbits out of five. Vitrectomy had a positive effect (p

PURPOSE To evaluate the concurrent rate of expression of c-Kit and its ligand stem cell factor (SCF) in uveal melanoma in respect to the further clinical course and the correlation of these markers. METHODS Paraffin sections from 35 primary uveal melanomas were evaluated immunohistochemically for the expression of c-Kit and SCF. Sixteen cases developed systemic metastasis during the follow-up (median: 5 years after diagnosis). The patients who did not develop metastasis (n = 19) had a mean follow-up of 10.6 (9-13) years. Radiation was performed in 6 patients. RESULTS c-Kit and SCF were expressed in all patients who did or did not develop metastasis in the further clinical course. A mean SCF expression of 77.2% (range 52.7%-97.5%) of tumors that did not develop systemic metastasis and 30.1% (range 2.9%-61.5%) of tumors with systemic metastasis were evident. Uveal melanomas revealing an increased SCF expression were found to develop no metastasis more frequently (p

Background Extracellular matrix remodelling regulated by matrix-metalloproteinase (MMP) inducer (CD147) is a crucial process during tumor cell invasion and regulation of blood supply. In this study, we evaluated the correlation of CD147 and MMP-2 expression with major prognostic factors for uveal melanoma and the development of metastasis. Methods The expression of CD147 and MMP-2 was analyzed in 49 samples of uveal melanomas. Triple immunofluorescence stainings using markers against glial cells (GFAP), endothelial cells (CD34) and macrophages (CD68) were performed to further analyse the exact localisation of CD147 and MMP-2 positivity. In 28 cases clinical metastatic disease were found. The remaining 21 cases showed no signs of metastatic disease for an average follow-up of 10 years. Correlation analysis (Pearson correlation) was performed to analyse the association of CD147 and MMP-2 expression with known prognostic factors, vasculogenic mimicry (VM), the mature vasculature (von Willebrand Factor) and tumor induced angiogenesis (by means of Endoglin expression). Results CD147 and MMP-2 were expressed in 47 (96.0 %) of the uveal melanomas. CD147 up-regulation was significantly correlated with a higher MMP-2 expression. The overall expression analysis revealed no significant difference in the metastatic (p = 0.777) and non-metastatic subgroup (p = 0.585). No correlation of CD147 expression and any system of blood supply was evident. In the non-metastatic sub-group a significant correlation of clustered CD147 positive cells with largest basal diameter (p = 0.039), height (p = 0.047) and TNM-stage (p = 0.013) was evident. Conclusions These data may indicate that CD147 regulates MMP-2 expression in uveal melanoma cells.