Sebastian Thaler

Decellularized Bovine Corneal Posterior Lamellae as Carrier Matrix for Cultivated Human Corneal Endothelial Cells

Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet’s membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), natrium hydrogen carboanhydrase (Na+/HCO3−), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry. Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet’s membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm2 on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na+/HCO3−, and Na+/K+-ATPase. Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.

Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage

Purpose:  To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N‐methyl‐D‐aspartate (NMDA)‐induced RGC damage.

In vivo toxicity study of rhodamine 6G in the rat retina.

PURPOSE To investigate the intraocular effect of rhodamine 6G (R6G) on retinal structures and function in an in vivo rat model and to develop an in vivo method for accurate evaluation of new dyes for intraocular surgery. METHODS R6G in physiologic saline solution (PSS) was injected into the vitreous of adult Brown Norway rats at concentrations of 0.0002%, 0.002%, 0.02%, 0.2%, and 0.5%. Control animals received only PSS. Retinal toxicity was assessed by retinal ganglion cell (RGC) counts, light microscopy 7 days later, photopic electroretinography (ERG), and measurement of scotopic sensitivity and recovery of dark adaptation 48 hours and 7 days after intravitreous injection. RESULTS R6G at concentrations of 0.2% and 0.5% led to a dose-dependent loss of RGC. The most significant loss occurred at 0.5%. Lower concentrations (0.0002%, 0.002%, and 0.02%) produced no statistically significant retinal ganglion cell loss. Analysis of the eyes by light microscopy showed no structural changes in the central retina, although injections of 0.5% R6G were followed by impressive degenerative changes adjacent to the injection sites. ERGs showed no effects of the highest R6G concentration on rods, kinetics of rhodopsin recovery after bleaching, or cone-driven responses. CONCLUSIONS R6G can be safely injected in doses of up to 0.02% in rats, but has a toxic effect on retinal ganglion cells at higher concentrations. Accumulation of R6G may be a problem at higher concentrations, particularly at the injection site.

Toxicity Study of Erucylphosphocholine in a Rat Model

Purpose: To investigate the effect of intraocular erucylphosphocholine (ErPC) on the retina, the retinal pigment epithelium (RPE), and the choroid in an in vivo rat model. Methods: Adult male Brown Norway rats were injected intravitreally with ErPC dissolved in balanced salt solution (BSS) at a final concentration of 10 or 100 μ M with BSS serving as control. Adverse effects on the anterior and posterior segment were assessed by slit-lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by electroretinography (ERG), retinal ganglion cell (RGC) quantification, and histology 7 days after intravitreal administration of ErPC. Results: There was neither a statistically significant difference in the clinical examination nor in the ERG waves of treated versus control rats 7 days after intravitreal administration of ErPC. Correspondingly, the number of RGC after BSS injection did not differ significantly from ErPC-injected animals. Histologic sections of the posterior segment of 10 and 100 μ M ErPC-injected rats did not show any signs of retinal toxicity. Electron microscopy did not display a difference between the 10 μM and the control group. Only the 100 μM-injected animals showed a discrete irregularity of the Müller cell and the retinal ganglion cell cytoplasm at the ultrastructural level. Conclusions: ErPC can safely be injected into the vitreous of adult rats at a concentration of 10 μM without any retinal toxicity. Even a 10-fold increase in ErPC concentration leads only to a discrete cytoplasmic irregularity of the innermost retinal layers.

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load.

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5-8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

Experimental evaluation of aniline and methyl blue for intraocular surgery.

Purpose: The purpose of this study was to investigate the biocompatibility of aniline and methyl blue in a well-established cell culture model and assess the staining properties of these dyes at the level of the internal limiting membrane (ILM) in human donor eyes. Methods: Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (ARPE-19 and primary human retinal pigment epithelium) cell proliferation. Cell viability was also quantified based on a two-color fluorescence assay (Life–Dead Assay). Aniline blue and methyl blue at a concentration of 0.2% was applied over the macula during vitrectomy in human donor eyes to evaluate the staining properties at the level of the ILM. Results: Both dyes and dye concentrations of 0.1% and 0.2% showed no toxic effect on ARPE-19 and primary human retinal pigment epithelium cell proliferation for exposure times of 1 and 10 minutes, respectively. Cell viability was also not affected at all. Both dyes provided a good contrast at the level of the ILM and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. Conclusion: Our results indicate that aniline blue and methyl blue might be applicable for intraocular surgery, providing a very good biocompatibility and required selective staining characteristics at the level of the ILM.

Intermediate uveitis and arthralgia as early symptoms in Whipple's disease.

A 56-year-old man presented at our clinic for a second opinion regarding a diagnosis of bilateral intermediate uveitis that had resisted treatment with local and systemic corticoids for three years. The patient had recurrent episodes of red eyes and blurred vision, predominantly in the right eye. All previous diagnostic tests were unrevealing. His medical history included arthralgia, which is not normally related to intermediate uveitis, and cataract surgery on the right eye several years earlier. No history of intestinal disorders, weight loss, or fever was reported. At our first examination visual acuity was 10/100 in the right eye and 10/20 in the left eye. Many white fluffy aggregates were present in the anterior chamber (Fig. 1) and vitreous body of the right eye, along with macular edema; however, there were no signs of retinal vasculitis. The left eye showed only mild inflammation. Chest radiography showed no signs of sarcoidosis or tuberculosis. Magnetic resonance imaging of the brain was unremarkable. Under suspicion of prolonged low-grade endophthalmitis, a diagnostic vitrectomy with lentectomy was performed, and white precipitates were collected for further analysis. Surprisingly, PCR analysis of these samples revealed Tropheryma whipplei DNA, leading to the diagnosis of ocular Whipple’s disease (WD). The patient then underwent a duodenal biopsy, which showed macrophages with periodic acid–Schiff-positive staining (Fig. 2). PCR analysis of this biopsy specimen and of blood

Caspase inhibitors protect against NMDA-mediated retinal ganglion cell death

Background:  Apoptosis is a major mechanism of cell death in glutamate‐induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA).

Neuroprotective effects of tempol acyl esters against retinal ganglion cell death in a rat partial optic nerve crush model.

Purpose:  The aim of this study is to search for more effective derivatives of the superoxide dismutase mimetic tempol (4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). Although tempol is neuroprotective in a rat partial optic nerve crush (PONC) model, relatively high doses are required to exert this effect.

In vivo toxicity testing of methyl blue and aniline blue as vital dyes for intraocular surgery.

Purpose: To investigate the biocompatibility of methyl blue and aniline blue as vital dyes for vitreoretinal surgery in an in vivo rat model and to evaluate the effect of these dyes on retinal structure and function. Methods: Adult Brown–Norway rats received intravitreal injections of 0.1%, 0.2%, and 2% methyl blue or aniline blue dissolved in balanced salt solution with balanced salt solution serving as a control. Retinal toxicity was assessed 7 days thereafter by means of retinal ganglion cell counts, light microscopy, and electroretinography. Results: No significant decrease in retinal ganglion cell counts at concentrations up to 0.2% was observed. At 2%, however, a significant retinal ganglion cell loss was detected with both dyes (more pronounced for aniline blue). Light microscopy showed no structural changes in the central retina for concentrations up to 0.2%. Electroretinographies detected no adverse effects of methyl blue or aniline blue on rod- or cone-driven responses at concentrations up to 0.2%. Conclusion: Methyl blue and aniline blue are very biocompatible and may, therefore, be usable for intraocular surgery. Further testing with other animal models will be necessary to confirm this. The safety margin of methyl blue is possibly higher than that of aniline blue.