Kai Januschowski

The retinal tolerance to bevacizumab in co‐application with a recombinant tissue plasminogen activator

Aim: To investigate the retinal toxicity of bevacizumab in co-application with a commercially available recombinant tissue plasminogen activator (rt-PA), and to facilitate a new therapeutic concept in the treatment of massive subretinal haemorrhage caused by neovascular age-related macular degeneration (AMD). Methods: Isolated bovine retinas were perfused with an oxygen-preincubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. Bevacizumab (0.25 mg/ml) and rt-PA (20 μg/ml) were added to the nutrient solution for 45 min. Thereafter, the retina was reperfused for 60 min with normal nutrient solution. Similarly, the effects of rt-PA (20 μg/ml, 60 μg/ml and 200 μg/ml) on the a- and b-wave amplitudes were investigated. The percentages of a- and b-wave reduction during application and at washout were calculated. Results: During application of bevacizumab (0.25 mg/ml) in co-application with 20 μg/ml (rt-PA), the ERG amplitudes remained stable. The concentrations of rt-PA alone (20 μg/ml and 60 μg/ml) did not induce significant reduction of the b-wave amplitude. In addition, 20 μg/ml rt-PA did not alter the a-wave amplitude. However, 60 μg/ml rt-PA caused a slight but significant reduction of the a-wave amplitude. A full recovery was detected for both concentrations during the washout. At the highest tested concentration of 200 μg/ml rt-PA, a significant reduction of the a- and b-wave amplitudes was provoked during the exposure. The reduction of ERG amplitudes remained irreversible during the washout. Conclusion: The present study suggests that a subretinal injection of 20 µg/ml rt-PA in co-application with bevacizumab (0.25 mg/ml) for the treatment of massive subretinal haemorrhage seems possible. This is a safety study. Therefore, we did not test the clinical effectiveness of this combined treatment.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells

Background/aims Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. Methods The effects of aflibercept (0.125, 0.5, 2 mg), ranibizumab (0.125 mg) and bevacizumab (0.3125 mg) after 1, 24, 48 and 72 h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. Results Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. Conclusions In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.

Rate and timing of spontaneous resolution in a vitreomacular traction group: Should the role of watchful waiting be re-evaluated as an alternative to Ocriplasmin therapy?

Introduction The incidence of spontaneous resolution of vitreomacular traction (VMT) is low in studies of Ocriplasmin that have had a limited follow-up. Previous studies did not look for morphological parameters in the natural history using spectral-domain ocular coherence tomography (SD-OCT) imaging. The purpose of this study was to investigate how often and when spontaneous VMT resolution occurs in candidates for Ocriplasmin therapy. Methods The study is a retrospective chart review of patients who would have high chances of a benefit by an Ocriplasmin injection, without epiretinal membrane or vitreomacular adhesion of 1500 µm or more on SD-OCT. Main outcome measures were the frequency of complete VMT resolution and the best corrected visual acuity seen in the natural history. Results Out of the 46 patients that were included after screening 889 SD-OCT images, 20 were found to exhibit spontaneous resolution during the follow-up period (median: 594 days, 95% CI 567 to 719 days), the majority after 6–12 months of observation (95% CI 266 to 617 days). The group with spontaneous VMT resolution and a mean improvement of one line in best corrected visual acuity included a few patients losing vision by macular hole formation. In the absence of resolution, patients lost on average one early treatment diabetic retinopathy study letter per year. Younger age was found to increase the chance of spontaneous resolution. Conclusions A shorter follow-up might underestimate the incidence of spontaneous VMT resolution as the functional outcome of watchful waiting. The likelihood of resolution does not seem to decrease after 12 months.

Immunomodulatory therapy with tumour necrosis factor α inhibitors in children with antinuclear antibody-associated chronic anterior uveitis: long-term results

Aim The aim of the study was to assess the long-term efficacy and tolerability of tumour necrosis factor α (TNFα) inhibitors in the therapy of children with refractory antinuclear antibody (ANA)-associated chronic anterior uveitis. Methods Retrospective analysis of 31 children with ANA-associated uveitis, treated with TNFα inhibitors with a follow-up period of at least 2 years. The outcome measures included: control of inflammation, corticosteroid-sparing potential and side effects. Results Twenty-three children (74%) were treated with adalimumab, five children (16%) with infliximab and three children (10%) with etanercept. Control of uveitis, defined as 0 anterior chamber cells while on ≤2 drops/day topical corticosteroids, was achieved in 22 of 31 patients (71%) after 1 year (95% CI 52% to 86%), and in 21 of 29 patients (72%) after 2 years of treatment (95% CI 53% to 87%). Control of uveitis was observed in 18 of 23 children (78%) treated with adalimumab, and in two of five children (40%) treated with infliximab. In all children treated with etanercept, no sufficient inflammatory control was found. Systemic corticosteroids could be discontinued in 71% (12/17 children) and topical corticosteroids in 55% (17/31) of the patients. Treatment-related side effects were found in nine children (29%, rate: 0.10/patient-year). Conclusions Our data show that adalimumab and infliximab have beneficial effects in the therapy of severe ANA-associated anterior uveitis in children.

The effects of triamcinolone crystals on retinal function in a model of isolated perfused vertebrate retina

A good clinical experience of intravitreal triamcinolone acetonide (TA) has been reported in several studies, but there are growing indications that epiretinal crystals of TA exhibit retinal toxicity. To investigate the effects of TA on retinal function we used a model of an electrophysiological in vitro technique for testing retinal toxicity. Isolated bovine retinas were perfused with an oxygen saturated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. After reaching stable ERG-amplitudes TA at the maximum solubility equilibrium (36 microg/ml) was either applied to the nutrient solution for 45 min or TA was administered epiretinally at concentrations (1 mg/ml, 4 mg/ml, 8 mg/ml, 20 mg/ml and 40 mg/ml) above the maximum solubility equilibrium to assure direct contact of the TA crystals with the isolated perfused retinas. After that the retinas were reperfused for 75 min with the standard nutrient solution. The percentage of a- and b-wave reduction directly after the application and at the washout was calculated. To assess the effects of TA at the level of the ganglion cell layer a Viability/Cytotoxicity Kit for mammalian cells was used. No changes of the ERG-amplitudes were detected during the exposure to 36 microg/ml TA for 45 min (b-wave: 9.6 microV+/-2.1 vs. 8 microV+/-2.1 (p=0.135); a-wave: -11 microV+/-2.7 vs. -10.6 microV+/-2.3 (p=0.889)) and at the washout (b-wave: 8 microV+/-2.1 vs. 8.3 microV+/-2.4 (p=0.18); a-wave: -10.6 microV+/-2.3 vs. -12 microV+/-2.6 (p=0.225)). At concentrations higher than 1mg/ml TA induced a decrease of the a- and b-wave in a concentration dependent manner. These changes were reversible for concentrations of TA up to 20mg/ml (b-wave: 9 microV+/-2.4 vs. 6.6 microV+/-2.5 (p=0.08); a-wave: -11.4 microV+/-2.0 vs. -11.2 microV+/-2.2 (p=0.37)), but irreversible at 40 mg/ml even at the end of the washout (b-wave: 9.8 microV+/-1.9 vs. 3 microV+/-1.7 (p=0.009); a-wave: -9.8 microV+/-2.1 vs. -2.6 microV+/-2.1 (p=0.001)). Histological examination of the preparations revealed a dramatic ganglion cell death, in which an application of 20mg/ml and 40 mg/ml TA led to a 60.53% (p=0.013) and 82.35% (p=0.002) ganglion cell death, respectively. The epiretinal application of 4 mg/ml TA and higher resulted in distinct effects on the ERG of the isolated perfused retinas. Ganglion cell death was induced at a concentration of 20mg/ml and higher. TA shows an asymmetric and partly high concentrated distribution after intravitreal application. Therefore, we consider concentrations of 4 mg/ml and higher might be toxic and should be avoided in clinical use.

Investigating the biocompatibility of two new heavy intraocular dyes for vitreoretinal surgery with an isolated perfused vertebrate retina organ culture model and a retinal ganglion cell line.

BackgroundDuring vitreoretinal surgery, vital dyes are used to visualize anatomical structures. Substances with a density higher than water are added to facilitate sedimentation and staining. BBG with 4% PEG (ILM Blue) and BBG with TB and 4% PEG (MBB Dual) are two new dyes. This study evaluates biocompatibility of the new dye solutions, using cell cultures and electrophysiological evaluation.MethodsTo determine cytotoxicity of ILM Blue and MBB Dual for 30, 60, 120 and 320 seconds, monolayer cultures of retinal ganglion cells (RGC5) were used. For functionality testing, bovine retinas were isolated and superfused with an oxygen-saturated nutrient solution, and the electroretinogram (ERG) was recorded. The two dye solutions were applied epiretinally for 30, 60 and 120 seconds. ERG recovery was monitored.ResultsAfter staining with ILM Blue, no statistical significant reduction of a- or b-wave amplitudes at the end of the wash-out was recorded. For MBB Dual, only a significant reduction of the a-wave amplitudes after 30 seconds of application at the end of the wash-out was noticed, while no statistically different changes for a- and b-wave amplitudes up to 120 seconds were noted. During the MTT assay, we noted no significant difference in cell viability after 30, 60, 120 and 320 seconds of staining with ILM blue, MBB Dual or 4% PEG in comparison to the control group (DMEM, Triton X-100 0.9% as positive control) after formazan extraction.ConclusionsILM Blue and MBB Dual seem to be safe for clinical use for a staining period of up to 120 seconds, probably even up to 320 seconds.

Measuring wearing times of glasses and ocular patches using a thermosensor device from orthodontics

Purpose:  Amblyopia is one of the most common visual disorders in children. The risk of severe visual impairment on the healthy eye is doubled in patients with amblyopia. If detected early enough, the chances of visual rehabilitation are good. Treatment consists of refractive correction and occlusion of the dominant eye. Patient compliance is an important factor and can be monitored using thermosensors. It was the goal of our study to give proof of the principle that the wearing times of glasses and patches can be measured using a comparatively small and commercially available microsensor.

Associating the magnitude of relative afferent pupillary defect (RAPD) with visual field indices in glaucoma patients

Purpose To identify the variable with the strongest association between the magnitude of the relative afferent pupillary defect (RAPD) and visual field indices in patients with glaucomatous optic neuropathy. Methods Seventy-nine consecutive subjects with manifest glaucomatous optic neuropathy at least in one eye were enrolled in this retrospective study. RAPD was assessed with the swinging flashlight test and quantified with a neutral density filter. Perimetry was performed using the fast thresholding strategy German Adaptive Threshold Estimation. The values of the central differential luminance sensitivity (DLS), of the MD (mean defect) and of the ‘loss volume’ (LVOL) based on the individually modelled 3D hill of vision—the latter two within the eccentricities of 10°, 20° and 30°, respectively—were entered into a linear regression model without intercept as a function of RAPD. Results An absolute value of RAPD of 0.3 log10 units or more was present in 20 out of 79 glaucoma subjects (25%). The magnitude of RAPD was most closely associated with LVOL-30° (R2=0.77), followed by MD-30° (R2=0.73), MD-20° (R2=0.71), LVOL-20° (R2=0.67), MD-10° (R2=0.58), LVOL-10° (R2=0.54) and central DLS (R2=0.04). Conclusions The prevalence of RAPD in glaucoma patients is comparatively small (25%). The magnitude of RAPD in glaucoma subjects is associated most closely with the LVOL within 30° eccentricity (which is the maximum visual field region tested in this study) and most loosely with central DLS, underscoring the impact of the entire (30°) visual field area on the afferent pupillary system.

Preservative-free triamcinolone acetonide injectable suspension versus "traditional" triamcinolone preparations: impact of aggregate size on retinal biocompatibility.

Purpose: To evaluate the biocompatibility of the three currently most commonly used triamcinolone acetonide (TA) preparations on retinal cells. Methods: Preservative containing KL (Kenalog-40; Bristol-Myers Squibb, Princeton, NJ), compounded preservative-free triamcinolone acetonide (PFTA; compounded from Volon A; Dermapharm, Vienna, Austria), and preservative-free triamcinolone acetonide injectable suspension (TRIESENCE; Alcon, Inc, Fort Worth, TX) (0.01–1 mg/mL) were either added directly on top or separated by a Boyden chamber filter or by a layer of vitreous to confluent cell cultures of retinal pigment epithelial cells (ARPE19) or retinal ganglion cells (RGC5). The distribution pattern of the TA crystals was assessed microscopically. Cell viability was assessed using MTT-ELISA and Live/Dead-Assay. Results: Sedimentation of triamcinolone acetonide injectable suspension, KL, or PFTA caused a pronounced decrease in cell viability. Cytotoxicity was most pronounced when triamcinolone acetonide injectable suspension and PFTA were used. Without direct sedimentation of TA crystals on top of the cells, none of the three formulations were cytotoxic. Triamcinolone acetonide injectable suspension showed the largest and most dense TA crystal aggregates on top of the cells. Conclusion: Retinal cytotoxicity of TA seems only to occur when there is intimate contact of TA crystals with the cellular membrane. Cytotoxicity depends on the number and size of TA crystal aggregates—with larger conglomerates being more harmful. Of the TA formulations tested, triamcinolone acetonide injectable suspension had the strongest tendency to form large TA crystal conglomerates and to gravitate downward.

The effects of ranibizumab (Lucentis) on retinal function in isolated perfused vertebrate retina

Background: Intraocular ranibizumab (Lucentis, Novartis, Basel Switzerland) is the primary choice in the treatment of neovascular age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is known to be a survival factor for neuronal cells. Therefore, blockage of all VEGF isoforms by ranibizumab could induce retinal dysfunction. Methods: Using isolated bovine retinas, the electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes, while the retinas were perfused with an oxygen preincubated nutrient solution. For 45 min, ranibizumab was applied at a concentration of 0.2 mg/ml and alternatively the solvent carrier without the active agent. The ERG was monitored before, during and after exposure. Results: The concentration of 0.2 mg/ml ranibizumab induced a non-significant b-wave reduction of 22.32% after exposure (p = 0.13). For the a-wave amplitude only a reduction of 4% was detected (p = 0.18). The solvent carrier induced no significant reduction of the a- and b-wave amplitudes (p = 0.30 and p = 0.979, respectively). Conclusion: In the ex vivo model, the isolated perfused vertebrate retina, ranibizumab has been proven to be a safe compound at the concentrations applied. The stability of the ERG-amplitudes rules out a considerable retinal dysfunction after an injection of up to 1 mg ranibizumab.