Johanna Hofmann

Novel maneuver facilitating Descemet membrane unfolding in the anterior chamber.

Purpose: To describe a new technique for unfolding of the Descemet membrane (DM) during Descemet membrane endothelial keratoplasty. Methods: DM and endothelium were stripped from donor corneas submerged in corneal storage solution. Donor DM diameters were 8.5 mm. The central 8 mm of DM was stripped from the recipient cornea. After staining with trypan blue to improve visualization, donor DM was inserted through a 2.75-mm incision. Unfolding was achieved with simultaneous digital pressure in the equatorial region and tapping of the corneal surface. This technique was performed in Descemet membrane endothelial keratoplasty patients with Fuchs endothelial dystrophy (n = 6) and bullous keratopathy (n = 4). Results: In all cases, a fast attachment of the DM was achieved. The recurling was successfully hindered because of the simultaneous equatorial pressurization and induced fluid currents inside the anterior chamber by appropriate corneal tapping. In all cases, a rapid corneal clearance was achieved with significantly increased visual acuity (logarithm of the minimal angle of resolution visual acuity preoperative = 0.90, logarithm of the minimal angle of resolution visual acuity final assessment = 0.19, P < 0.001; follow-up 14 ± 5 weeks). Conclusion: Simultaneous digital pressure with corneal tapping is a helpful maneuver to unfold the DM inside the anterior chamber. This technique may reduce the need for unnecessary instrumental and noninstrumental manipulation of the graft.

Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts

Purpose:  To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering.

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.

Comparison of pneumatic dissection and forceps dissection in Descemet membrane endothelial keratoplasty: histological and ultrastructural findings.

Purpose: To compare air and forceps dissection of Descemet membrane (DM) in regard to endothelial cell loss, apoptosis, and ultrastructural findings for transplantation in Descemet membrane endothelial keratoplasty. Methods: DM of 32 corneoscleral rims were separated using forceps dissection (group A) and air dissection (group B) and had been organ cultured for 1 week. The endothelial cell density (ECD) was evaluated before and immediately after DM dissection and during 1 additional week of organ culture. The presence of any residual stroma, thickness of the DM, thickness of banded and nonbanded layers, and smoothness of the lamella were analyzed. Apoptosis was evaluated by TUNEL (terminal dUTP nick-end-labeling) staining. Results: In group A, 15 of 16 usable DM were available, and in group B 14 of 16 usable DM were available. The duration of preparation was 19.7 minutes in group A and 8.8 minutes in group B. The mean ECD was 2050 ± 167 cells per square millimeter in group A and declined to 1846 ± 153 cells per square millimeter after 1 week of organ culture. The mean ECD in group B was 2038 ± 212 cells per square millimeter and declined to 1863 ± 211 cells per square millimeter. No evidence of adherent rests of corneal stroma in any specimens in either group was observed, demonstrating a stroma free separation with no differences in the smoothness of the lamellae. The rate of apoptosis positive cells was 0.37% in group A and 0.40% in group B. Conclusions: Both separation methods seem to be equal in regard to the quality of the obtained DM. Finally, it depends on the preference of the surgeon which dissection method to use.

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.

Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model.

Purpose:  To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model.

Histological and ultrastructural findings of corneal tissue after failed descemet membrane endothelial keratoplasty

Purpose:  To investigate in a retrospective review the histological and ultrastructural findings after failed primary and early Descemet membrane endothelial keratoplasty (DMEK), propose possible pathomechanisms of graft failure and give clinical implications.

Comparison of Swollen and Dextran Deswollen Organ- Cultured Corneas for Descemet Membrane Dissection Preparation: Histological and Ultrastructural Findings

PURPOSE To compare the use of swollen tissue versus tissue deswollen by addition of dextran to the medium in the dissection of organ-cultured Descemet membrane (DM) with regard to preparation-related characteristics and ultrastructural findings to optimize transplantation in Descemet membrane endothelial keratoplasty (DMEK). METHODS DMs of 20 corneoscleral rims were separated using organ-cultured groups, one immersed in culture medium without dextran (group A) and the other in medium with added dextran for 24 hours (group B). The preparation details were noted. The difficulty of preparation was analyzed using a scoring system (0 = impossible, 10 = easy). By means of a micrometer, ultrathin sections of endothelial layer were obtained. Presence of any residual stroma, thickness of the DM, thickness of the endothelial cell layer, and the smoothness of the lamella were analyzed. RESULTS In both group A and group B, all 10 usable DMs were available. Mean preparation time was 6.5 ± 1.4 minutes in group A and 6.1 ± 0.9 minutes in group B (P = 0.399). The difficulty score was 7.9 ± 1.9 in group A and 8.0 ± 2.0 in group B (P = 0.726). The total mean thickness of the DM (without the endothelial cell layer) was 13.58 ± 2.81 μm in group A and 12.69 ± 2.06 μm in group B (P = 0.474). The total mean thickness of the endothelial cell layer was 3.99 ± 0.62 in group A and 3.98 ± 0.52 in group B (P = 0.989). Light microscopy and transmission electron microscopy revealed no evidence of any adherent remnants of corneal stroma on any specimen in each group. CONCLUSIONS DM-endothelium grafts for transplantation in DMEK procedures can be surgically prepared from organ-cultured corneal rims in swollen and deswollen conditions. Both separation methods seem to be equivalent in regard to the preparation characteristics of the obtained DM. Nevertheless, clinical studies are still necessary in order to confirm these findings.

Trichostatin A induces cell death at the concentration recommended to differentiate the RGC-5 cell line.

Supplementation with Trichostatin A (TSA) has been described as the method of choice for differentiating the RGC-5 cell line into cells with neuronal properties. However, TSA is known to induce apoptosis. We therefore investigated whether TSA at the recommended concentration for differentiation (500 nM) and at three additional concentrations (40, 150 and 2000 nM) induces apoptosis or cell death in the RGC-5 cell line. Morphological changes of the RGC-5 cells occurred after 24 and 48 hours (h) of treatment with 500 and 2000 nM TSA. Differentiation of RGC-5 cell began at 150 nM. A decrease in the cell count was observed from 150 nM TSA onwards compared to controls. Five hundred nanomolar of TSA reduced the amount of cells to 51% (p<0.005) after 24h and to 24% (p<0.005) after 48 h compared to controls on crystal violet staining. At 500 nM TSA a massive induction of apoptosis after 24 and 48 h was noted. Supplementation of 500 nM TSA increased caspase 3/7 activity 5.0-fold (p<0.005). Furthermore, 27× more TUNEL-positive cells were found and the cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.1) higher 24h after the addition of 500 nM TSA. The Bax/Bcl-2 ratio was 3.4-fold (p<0.05) higher after 48 h. Cell viability decreased to 70% (p<0.005) and to 35% (p<0.005) after 24 and 48 h, respectively. Moreover, 103× (p<0.05) more dead cells (via propidium iodide staining) were found after 48 h of treatment with 500 nM TSA. In conclusion, TSA induces cell death and apoptosis at the concentration recommended for differentiation. The induction of apoptosis occurred dose and time dependently and already at even lower concentrations of TSA which did not lead to differentiation induced apoptosis. Thus, studies with RGC-5 cells should not be performed within the first 48 h after supplementation with TSA.

Cytotoxic Properties of Sunitinib and Sorafenib on Human Corneal Epithelial Cells

Abstract Purpose: To generate toxicology profiles of individual drugs on human corneal epithelial cells (HCEC) and compare their in vitro cytotoxicity. Methods: Monolayer cultures of HCEC were harvested from two human donor eyes. Sunitinib (0.3–10 µg/mL) and Sorafenib (0.3–100 µg/mL), diluted in culture medium (CnT-BM.1, CELLnTEC Advanced Cell Systems AG, Bern, Switzerland), 1% Penicillin and 1% Streptomycin were added to cells that were being grown in cell culture dishes. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed 24 hours, three days and five days after incubation. Live/dead viability/cytotoxicity assay (Live/dead assay) was performed and analyzed using fluorescence microscopy after 24 hours of incubation. The expression of p63, ABCG2 and PDGFRβ was evaluated by immunocytochemistry prior to exposure. Cell morphology was assessed with a phase contrast microscope after 24 hours of exposure. Results: Significant toxicity of Sunitinib was seen at concentrations of >3.3 µg/mL and of Sorafenib at concentrations of >1.0 µg/mL after 24 hours of incubation. Both drugs exhibited increasing toxicities over time. HCEC stained positively for p63, ABCG2 and PDGFRβ. In comparison, the IC50 (inhibitory concentration 50) of Sorafenib was 2.26 times the IC50 of Sunitinib using Live/dead assay after 24 hours and 2.39, 1.29 and 0.78 times the IC50 of Sunitinib using the MTT test after 24 hours, three days and five days, respectively. Conclusions: These in vitro experimental findings support the safety of Sunitinib and Sorafenib on HCEC when used at a concentration of <3.3 µg/mL and <1.0 µg/mL, respectively, after 24 hours of exposure. The in vitro cytotoxicity of Sorafenib on HCEC was higher than Sunitinib.