Julia Lüke

The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina.

Aims: To investigate the outcomes of Rho-kinase inhibition in the electrophysiological ex vivo model of the isolated perfused vertebrate retina under hypoxia. Methods: Bovine retinas were perfused with an oxygen saturated nutrient solution with or without the Rho-kinase inhibitor H-1152P. The retinas were stimulated repeatedly until stable amplitudes were reached and the electroretinogram was recorded at five minute intervals. Hypoxia was induced for 15, 30, and 45 minutes, after which the oxygen saturation was restored. The extent of the cell damage and glial reactivity was determined by Ethidium homodimer-1 staining, immunohistochemistry, and Western blot. Results: Hypoxia caused a time-dependent reduction of the b-wave amplitudes, which could not be prevented by the H-1152P. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes, these effects did not reach statistical significance. Hypoxia also resulted in an increase in cell damage and the activation of the glial cells in the untreated retinas whereas the administration of H-1152P significantly reduced the extent of these events. Conclusion: H-1152P exerted a neuroprotective effect against necrosis on the isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the inhibitor could not prevent the hypoxia induced retinal dysfunction possibly due to the interference with synaptic modulation.

A TGF-β receptor 1 inhibitor for prevention of proliferative vitreoretinopathy.

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 Tenons 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.

A p38 MAPK inhibitor improves outcome after glaucoma filtration surgery.

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 &mgr;M) 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 &mgr;M SB 202190, and G4=50 &mgr;M 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 &mgr;M). 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 &mgr;M 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.

The Concentration-Dependent Effects of Indocyanine Green on Retinal Function in the Electrophysiological ex vivo Model of Isolated Perfused Vertebrate Retina

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.

The Effect of Adjuvant Dimethylenastron, a Mitotic Kinesin Eg5 Inhibitor, in Experimental Glaucoma Filtration Surgery

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

The retinal biocompatibility of dyes in the ex vivo model of the isolated superfused vertebrate retina.

Background: Peeling of the internal limiting membrane or epiretinal membranes is a successful principle in macular surgery to achieve a functional benefit. Different dyes are used to facilitate the identification of intraocular tissues. The aim of our work was to investigate the retinal tolerance to the different dyes and their solvent carriers to provide valuable data for surgeons in handling for an optimal intraoperative use. Methods: Using the ex vivo model of the isolated superfused vertebrate retina technique, the effects of the dyes were tested on human and bovine retinal function. The retinas were perfused with an oxygen preequilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl electrodes. After recording stable ERG amplitudes, the dyes brilliant blue G, indocyanine green, trypan blue, patent blue, triamcinolone and their solvent carriers were investigated. Results: Reductions of the ERG amplitudes were found for each tested dye. The effects after application of the dyes were dependent on time and concentration of the applied dyes, which were different for each dye. Conclusion: In part, the ERG has shown strong effects already after a short period of dye application. Surgeons who rely on the intraocular use of the dyes should keep in mind our findings, and the use of some dyes should be limited to selected cases. The well-considered use of the dyes by the surgeons could lead to a better functional outcome and avoid a possible harmful outcome of the surgery after mishandling.

Expression of c-Kit and its ligand SCF in primary uveal melanoma

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<0.0001; hazard ratio (HR)=0.963, 95% confidence interval (CI) 0.945–0.981). A mean c-Kit expression of 58.01% (range 5.9%-97.9%) in the group who did not develop metastasis and 48.9% (range 5.0%-95.9%) in the group with systemic metastasis were observed. c-Kit expression was not associated with increased rates of metastasis formation (p=0.7329; HR=0.997, 95% CI 0.982–1.013). The correlation between SCF and c-Kit is weak (0.39; 95% CI 0.06–0.63). Conclusions c-Kit expression was not found to be associated with metastasis formation. A high SCF expression of primary choroidal melanomas was significantly associated with a lower incidence of systemic metastasis, which indicated SCF as a benign prognostic factor in the further clinical course.

Biocompatibility of the vital dye Acid Violet-17 on retinal pigment epithelial cells

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 <0.125 mg/mL is likely to be well tolerated by the RPE cells, whereas the concentrations from 0.25 mg/mL onward can reduce viability and induce dedifferentiation particularly after long-term exposure.

The effect of disease-modifying antirheumatic drugs on retinal function in the electrophysiological ex vivo model of the isolated perfused vertebrate retina.

Background: Disease-modifying antirheumatic drugs (DMARDs) have been suggested in the treatment of inflammatory ophthalmological diseases. The aim of the present study was to investigate the effects of these DMARDs on bovine retinal function. Methods: Bovine retina preparations were perfused with a standard solution. After recording stable electroretinograms the nutrient solution was substituted by a DMARD medium with varying concentrations of different drugs (etanercept and infliximab) for 30 min. Afterwards b-wave recovery was observed. Results: Significant decreases in the b-wave amplitude (p < 0.05) were found for etanercept 0.5 mg/ml (p = 0.0022). Infliximab 2 mg/ml (p = 0.1276) did not result in any statistically significant b-wave reduction. Conclusion: The presented data suggest that infliximab might have the better safety profile than etanercept.

Morphological and Functional Outcome after Brilliant Blue G-Assisted Macular Hole Surgery

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.