Jose Hurst

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.

Efficacy of two different thiol-modified crosslinked hyaluronate formulations as vitreous replacement compared to silicone oil in a model of retinal detachment

The efficacy of two novel artificial vitreous body substitutes (VBS) consisting of highly biocompatible thiolated cross-linked hyaluronic acid (HA)-based hydrogels in comparison to silicone oil in a model of retinal detachment was investigated. Pars plana vitrectomy (23G) was performed in the right eye of 24 pigmented rabbits. Retinal detachment of two quadrants was induced by creating a small retinotomy near the vascular arcade and injecting balanced salt solution (BSS) subretinally. The retina was reattached by injecting air, which was followed by increasing the infusion pressure, and the retinal tear was treated by endolaser photocoagulation. At the end of the procedure, the eye was filled either with 5000-cs silicone oil (after fluid air exchange) or the respective hydrogel (with two different viscosities). Follow-up examination included slit lamp examination, funduscopy, intraocular pressure measurements (IOP), optical coherence tomography (OCT) and electroretinogram (ERG) measurements. After a maximum follow-up of four weeks both eyes were removed, examined macroscopically, photographed, and prepared for histology. Of the eight rabbits that received silicone oil, seven (87.5%) developed a recurrent retinal detachment with pronounced proliferative vitreoretinopathy within the first two weeks after surgery. In contrast, in the hydrogel treated eyes, the retina stayed attached in the majority of the cases (73.3%). IOP and retinal morphology were normal as long as the retina remained re-attached. In conclusions, this model of retinal detachment, both thiolated crosslinked hyaluronate hydrogels showed superior efficacy when compared to silicone oil. These hydrogels have a promising potential as novel vitreous body substitutes.

Degenerative effects of cobalt-chloride treatment on neurons and microglia in a porcine retina organ culture model.

Abstract In order to understand the pathological processes of retinal diseases, experimental models are necessary. Cobalt, as part of the vitamin B12 complex, is important for neuronal integrity. However, it is known that high quantities of cobalt induce cytotoxic mechanisms via hypoxia mimicry. Therefore, we tested the degenerative effect of cobalt chloride (CoCl2) on neurons and microglia in a porcine retina organ culture model. Organotypic cultures of porcine retinas were cultured and treated with different concentrations of CoCl2 (0, 100, 300 and 500 &mgr;M) for 48 h. After four and eight days, CoCl2 induced a strong degeneration of the porcine retina, starting at 300 &mgr;M. A loss of retinal ganglion cells (RGCs, Brn‐3a), amacrine cells (calretinin) and bipolar cells (PKC&agr;) was observed. Additionally, a high expression of hypoxia induced factor‐1a (HIF‐1a) and heat shock protein 70 (HSP70) was noted at both points in time. Also, the Caspase 3 protein was activated and P21 expression was induced. However, only at day four, the Bax/Bcl‐2 ratio was increased. The effect of CoCl2 was not restricted to neurons. CoCl2 concentrations reduced the microglia amount (Iba1) and activity (Iba1 + Fc&ggr;‐Receptor) at both points in time. These damaging effects on microglia were surprising, since CoCl2 causes hypoxia and a pro‐inflammatory environment. However, high concentrations of CoCl2 also seem to be toxic to these cells. Similar degenerative mechanisms as in comparison to retinal ischemia animal models were observed. In summary, an effective and reproducible hypoxia‐mimicking organotypic model for retinal degeneration was established, which is easy to handle and ready for drug studies. HighlightsCoCl2 stabilized HIF‐1&agr; during the cultivation period.CoCl2 induced a neuronal degeneration of the porcine retina; starting at 300 &mgr;M.High concentrations of CoCl2 are generally toxic for neurons and microglia.Retinal degeneration occurred via apoptosis and cellular senescence mechanisms.The intrinsic pathway via Bax played a role only at the beginning of the cultivation.

Comparison of Different Cell Culture Media in the Model of the Isolated and Superfused Bovine Retina: Investigating the Limits of More Physiological Perfusion Solutions

ABSTRACT Purpose: The isolated superfused retina is a standardized tool in ophthalmological research. However, stable electroretinogram (ERG) responses can only be obtained for around eight hours; therefore, limiting its use. The aim of this study was to evaluate the short-term potential of different cell culture media and to promote long-term testing based on the results obtained. Materials and Methods: For the experimental procedure bovine retinae were prepared and perfused with the standard Sickel solution and an ERG was performed. After recording stable a- or b-waves, different media (Dulbecco’s Modified Eagle’s Medium (DMEM), MACS, and Neurobasal) were superfused for 45 minutes. ERG recovery was monitored overall for 75 minutes. Analysis of the mRNA expression of Thy-1, GFAP, Bax/Bcl-2-ratio, Rhodopsin, and Opsin via qRT-PCR was performed directly after ERG recording on the same retina. Results: None of the tested media had a negative effect on a-wave amplitudes, although b-wave amplitudes decreased (DMEM) or increased (MACS and Neurobasal) compared to the standard solution (Sickel) after 45 minutes of exposure. However, after 75 minutes of wash-out, no difference to the standard solution alone could be observed. Exposure to different media either had no effect or decreased the Opsin and Rhodopsin mRNA levels. Thy-1 expression was strongly diminished in DMEM and MACS (by 2–3-fold), whereas incubation in Neurobasal medium led to a slight increase compared to incubation with the standard solution. Furthermore, the Bax/Bcl-2 ratio indicated an anti-apoptotic effect (Bax/Bcl-2 = 0.16; p < 0.05) for Neurobasal. Conclusion: Neurobasal medium displayed the best electrophysiological properties in the short-term and may be applicable for stable long-term escalation testing.

Negative Effects of Acid Violet-17 and MBB Dual In Vitro on Different Ocular Cell Lines

ABSTRACT Purpose: Vital dyes have become a clinical standard during vitrectomy to visualize anatomical structures. It was the aim of this study to test the effect of two vital dyes (AV 17-M with 5% mannitol and MBB Dual) on different intraocular cells, to see whether in vitro test can be used as reliable preclinical testing tool. Methods: Cell morphology was assessed via phase contrast pictures, cell viability via MTS assay and total cell amount via crystal violet staining. ARPE19 and 661W cells were chosen for toxicology testing at different exposure times (60 seconds, 15 minutes and 30 minutes). Vital dyes were completely removed after the staining period. Results: Treatment with AV 17-M changed the morphology and the cell number at every time point investigated on ARPE19 and 661 W cells. ARPE19 cells treated with AV 17-M or MBB Dual displayed only a slight or no decrease in cell viability after the three different exposure times. AV-17 without medium to simulate a possible intraoperative use after fluid-air exchange showed a decrease in viability of 6%, 24% and 14%. A difference in cell density of 21%, 46% and 34% was noted after CV staining for AV 17-M, MBB Dual led to a decrease of 2%, 16% and 3% after 30 minutes compared to BSS. AV 17-M directly applied on 661W decreased viability significantly by 18% after 60 seconds, 33% after 15 minutes and 40% after 30 minutes. Cell density of 661W cells exposed relevant negative effects; after incubation of 60 seconds with AV 17-M, the cell amount was significantly lowered by 41% and MBB Dual by 12%. After 15 minutes, a loss of 48% cell amount was detected with AV 17-M and after 30 min 51%. MBB Dual led to 37% loss after 15 minutes and to 28% loss after 30 minutes. Conclusion: AV 17-M with 5% mannitol has a negative effect on different ocular cells.

Novel mouse model for primary uveal melanoma: a pilot study.

To establish a mouse model with the aim of studying the tumour biology and metastasis formation of uveal melanoma.

Characterization of a Standardized Ex-vivo Porcine Model to Assess Short Term Intraocular Pressure Changes and Trabecular Meshwork Vitality After Pars Plana Vitrectomy with Different Silicone Oil and BSS Tamponades

ABSTRACT Purpose: The aim of this study was to characterize a standardized porcine ex-vivo testing system for intraocular pressure (IOP) monitoring after vitrectomy with different endotamponades. Methods: Twenty-four pig eyes, six per endotamponade group were obtained immediately postmortem. After pars plana vitrectomy, vitreous substitutes (silicone oil 1000 mPas, 2000 mPas, 5000 mPas, and Balanced Salt Solution (BSS)) were instillated and IOP was observed over 24-hours. Infusion pumps with Dulbecco’s Modified Eagle Medium (DMEM) simulated a constant aqueous humor circulation. A histological examination of the trabecular meshwork with DAPI- and TUNEL-staining was performed to detect the amount of apoptotic cells. Results: TUNEL-assay showed a mean cell death rate of 3.78% (SD ± 1.46%) for silicone oil endotamponades compared to 5.05% (SD ± 2.18%) in BSS group. One-way ANOVA (p = 0.425) showed no significant difference between both groups. Mean IOP in silicone oil endotamponades was 9.50 mmHg (SD ± 1.68 mmHg) at baseline, 13.23 mmHg (SD ± 0.79 mmHg) after 1 hour, 18.46 mmHg (SD ± 2.13 mmHg) after 12 hours and 15.51 mmHg (SD ± 2.82 mmHg) 24 hours after instillation. A comparison of all silicone oil groups (one-way ANOVA, Bonferroni post-hoc test, p = 0.269 to 1.000) didn’t reveal significant differences in mean IOP. Conclusion: The standardized ex-vivo porcine model represents an effective alternative to the in-vivo testing in animals. Maintaining the trabecular and uveoscleral outflow pathway enables a pseudo in-vivo analysis.