Maximilian Schultheiss

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.

Biocompatibility and antifibrotic effect of UV-cross-linked hyaluronate as a release-system for tranilast after trabeculectomy in a rabbit model--a pilot study.

Purpose: To analyze the release kinetics and the clinical and histological effects of UV-cross-linked hyaluronic acid as a release-system for the transforming growth factor β-2 antagonist tranilast with anti-phlogistic properties on intraocular pressure after trabeculectomy in an aggressive scarring animal model. Methods: Hyaluronate acid was UV-cross linked and loaded with tranilast. The release of tranilast into a buffered salt solution was assessed spectrophotometrically. Glaucoma filtration surgery, similar to that performed in clinical practice, was performed on chinchilla rabbits. The rabbits were divided in 3 groups. (Group A: trabeculectomy alone, group B: trabeculectomy with a cross-linked hyaluronic acid gel preparation and group C: trabeculectomy with cross-linked hyaluronic gel preparation mixed with tranilast). Antifibrotic efficacy was established by clinical response and histologic examination. Results: The cross-linked gels released tranilast for up to 26 h. The release plotted as a function of the square root of time was consistent with a largely diffusion-controlled release system. Both the gel preparation alone and the gel preparation mixed with tranilast were well tolerated in vivo. No adverse effects such as inflammation, corneal toxicity or blurring of the optical media were observed. The intraocular pressure reached preoperative levels within 9 days after surgery in control animals and group B, but remained significantly reduced (p = 0.00016) in the group with tranilast until day 22. Conclusions: The data of this pilot study suggest that the intraoperative application of UV-crossed linked hyaluronic acid used as a slow release system for tranilast may improve the surgical outcome of glaucoma filtration surgery.

Neuroprotective effects of a taurine-containing irrigation solution for vitrectomy.

Background: During pars plana vitrectomy, the retina is exposed to several iatrogenic risk factors, including excitotoxicity. A taurine-containing irrigation solution for pars plana vitrectomy (PURI PROTECT) has been developed and is claimed to have neuroprotective properties. Methods: Retinal ganglion cells (RGC-5) and retinal whole mounts were incubated in standard irrigation solution (SIS) and SIS supplemented with 3 mM taurine (SIS-taurine). Excitotoxicity was induced by the addition of 8, 10, and 12 mM or 250 &mgr;M glutamate. Cell viability and cell survival were assessed by the MTT test and Annexin-V/propidium iodide flow cytometry. Whole mounts were stained with the Live/Dead staining assay. Pars plana vitrectomy with SIS or SIS-taurine was performed in rabbits. Animals were followed-up by electroretinography. Results: RGC-5 incubated in SIS-taurine showed a 4.3-fold (P < 0.0005) better overall cell viability and an up to 8.5-fold (P < 0.05) increased cell survival under excitotoxic conditions compared with that incubated in SIS. Whole mounts incubated in SIS-taurine showed a 1.7-fold (P < 0.0005) and 1.6-fold (P < 0.0005) better cell survival under excitotoxic and nonexcitotoxic conditions, respectively. In the immediate postoperative period, b-wave amplitudes were significantly better in animals operated with SIS-taurine compared with control (P < 0.01). Conclusion: A taurine-containing irrigation solution may protect retinal ganglion cells against excitotoxicity.

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia.

Purpose Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies. Methods Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed. Results Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia. Conclusion Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.

Glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing. Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 µm glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min. For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p<0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p=0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p<0.01, decrease of 25.5% at the end of the washout, p=0.03). For 250 µm glutamate, an initial 7.8% reduction of a-wave amplitudes (p>0.05) followed by a reduction of 1.9% (p>0.05). A reduction of 83.7% of b-wave amplitudes (p<0.01) was noted; after a washout of 75 min the reduction was 2.3% (p=0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

Pupillary light reaction during high altitude exposure.

Purpose This study aimed to quantify the pupillary light reaction during high altitude exposure using the state of the art Compact Integrated Pupillograph (CIP) and to investigate a potential correlation of altered pupil reaction with severity of acute mountain sickness (AMS). This work is related to the Tübingen High Altitude Ophthalmology (THAO) study. Methods Parameters of pupil dynamics (initial diameter, amplitude, relative amplitude, latency, constriction velocity) were quantified in 14 healthy volunteers at baseline (341 m) and high altitude (4559 m) over several days using the CIP. Scores of AMS, peripheral oxygen saturation and heart rate were assessed for respective correlations with pupil dynamics. For statistical analysis JMP was used and data are shown in terms of intra-individual normalized values (value during exposure/value at baseline) and the 95% confidence interval for each time point. Results During high altitude exposure the initial diameter size was significantly reduced (p<0.05). In contrast, the amplitude, the relative amplitude and the contraction velocity of the light reaction were significantly increased (p<0.05) on all days measured at high altitude. The latency did not show any significant differences at high altitude compared to baseline recordings. Changes in pupil parameters did not correlate with scores of AMS. Conclusions Key parameters of the pupillary light reaction are significantly altered at high altitude. We hypothesize that high altitude hypoxia itself as well as known side effects of high altitude exposure such as fatigue or exhaustion after ascent may account for an altered pupillogram. Interestingly, none of these changes are related to AMS.

Hypothermia Promotes Survival of Ischemic Retinal Ganglion Cells

PURPOSE Ischemic stroke in retinal arteries leads to death of neural tissue and ultimately to blindness. The retina is known to die within 4 hours after onset of ischemia. It is debated whether hypothermia might increase the time window for medical treatment and thereby the chance of recovering sight. In order to characterize the time course of cell death during ischemia and potential beneficial effects of hypothermia in more detail, we investigated the survival of ganglion cells in ischemic pig and human retina as a function of time and temperature. METHODS Eyes were obtained from minipigs and from human donors post mortem. Enucleated minipig eyes were stored for defined durations at three different temperatures (37 °C, 21 °C, and 4 °C). In order to assess the viability of the tissue, we measured ganglion cell activity (spiking) with multielectrode arrays. RESULTS Minipig retinal ganglion cell function was severely compromised after 2 hours of ischemia at body temperature. After 4 hours, ganglion cells did not fire action potentials anymore. However, at 21 °C, ganglion cell activity was maintained under ischemic conditions for up to 12 hours, and for at least 50 hours at 4 °C. In postmortem human retina, we recorded ganglion cell activity in retinas received up to 27 hours after death. CONCLUSIONS Our results demonstrate that hypothermia greatly increases survival of retinal ganglion cells exposed to ischemia. These results might be relevant for the future treatment of retinal ischemia.

Comparing the Effects of Two Different Irrigation Solutions on an Isolated Perfused Vertebrate Retina

Purpose: To compare the effect of a taurine-containing intraocular irrigation solution (PuriProtect™) to a standard irrigation solution (BSS™) we evaluated the retinal function using an electroretinogram (ERG) and analyzed the survival of retinal ganglion cells on isolated whole mount retinas. Materials and Methods: During ERG recordings, each irrigation solution was superfused for 45 min with the relevant irrigation solution. To investigate the effects on photoreceptor function, 1 mM asparate was added to obtain a-waves. The recovery of the a- and b-wave was monitored after superfusing the retinas with standard medium again. To evaluate the percentage of dead ganglion cells, retinas were stored for 24 h at 4°C in darkness and after staining the retinas with ethidium homodimer-1 the retinas were analyzed using fluorescence microscopy. Results: The application of standard medium supplemented with 2 mM taurine resulted in a significant increase of the b-wave amplitude compared to standard medium alone. The a-wave amplitudes showed no significant changes under taurine supplementation. Compared to standard medium BSS showed no significant decrease in b-wave amplitudes, but a significant decrease in a-wave amplitudes. In contrast to BSS there were no significant changes in the a- or b-wave amplitudes detectable after the application of PuriProtect. At the end of the washout period no significant changes in a- or b-wave amplitudes were recorded for any tested irrigation solution. Retinas stored for 24 h in PuriProtect or in standard medium with taurine had a statistically significant smaller amount of dead cells than retinas stored in standard medium without taurine supplementation. Conclusions: BSS does not seem to be an ideal irrigation solution, because it compromises the a-wave in the ERG. In contrast to BSS, PuriProtect showed no significant impact on the ERG and showed a better long-term effect on ganglion cell survival. Taurine supplementation, therefore, seems to be neuroprotective and its supplementation to an intraocular irrigation solution favorable for the retina.

α5β1-Integrin inhibitor (CLT-28643) effective in rabbit trabeculectomy model

Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5β1‐integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5β1‐integrin inhibitor (CLT‐28643).

Cyclosporine a protects RGC-5 cells from excitotoxic cell death.

Purpose:Cyclosporine A (CSA) is a widely used immunosuppressive drug. Furthermore, CSA showed neuroprotective properties in several neurological disorders. However, nearly no data exist regarding CSA and its possible neuroprotective effect on retinal ganglion cells (RGCs). Methods:RGC-5 cells were stressed with 10 mM glutamate for 24 hours with or without adding varying concentrations of CSA (1, 3, 6, or 9 &mgr;g/mL) to the medium. Cell viability and cell density were analyzed by MTS assay and crystal violet staining, respectively. Induction of apoptosis was determined through caspase 3/7 activity and Annexin V+/PI− flow cytometry. Results:The incubation of RGC-5 cells with 10 mM glutamate for 24 hours induced a 3.1-fold and a 3.4-fold decrease in overall cell viability and cell density, respectively, compared with controls. The supplementation of 9 &mgr;g/mL CSA to 10 mM glutamate led to a 2.7-fold increase in overall cell viability (P<0.0005) and a 2.5-fold increase in cell density (P<0.0005) compared with RGC-5 cells treated only with 10 mM glutamate. Furthermore, the addition of 9 &mgr;g/mL CSA to 10 mM glutamate significantly reduced caspase 3/7 activity by 1.3-fold (P<0.0005) and the amount of Annexin V+/PI– cells by 2.8-fold compared with RGC-5 cells incubated with 10 mM glutamate alone. The neuroprotective effect of CSA dose-dependently decreased with lower concentrations. Conclusions:CSA can effectively protect RGC-5 cells against glutamate-induced excitotoxicity. Therefore, CSA should be tested in further experiments to evaluate its potential as a neuroprotective substance against RGC disorders.