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Purpose: To evaluate the efficiency of cultivated human corneal endothelial cells (HCECs) to dehydrate the cornea, using models of the posterior cornea, composed of artificial collagen mass (to represent corneal stroma) and equine collagen membranes (to represent Descemet membrane)., Methods: HCECs were isolated from donor corneal rings and cultivated at 37°C in 5% CO2 and 95% humidified air. The study design included 4 different sets of models: in set 1, the HCECs were placed directly on the collagen mass complex; in set 2, HCECs were placed on a thin equine collagen membrane and laid over the collagen mass; in set 3, HCECs were placed on a thick equine collagen membrane laid over the collagen mass; and in set 4 (the control group), the hydrophilic collagen mass was left alone to interact with the nutritional medium. The minimum thickness of each sample was measured with optical coherence tomography directly before placement of cells and after exposure to the nutritional fluid for 48 hours., Results: After 2 days of exposure to the nutritional medium, the percentage decreases in thickness in "posterior cornea" models were 66% for set 1, 57% for set 2, and 13% set 3. In the control set, measurement of thickness after 2 days of exposure was not possible because of excessive fluid absorption., Conclusions: This in vitro study of HCECs showed that the dehydrating ability of HCECs is adversely affected by increased thickness of the artificial (Descemet) membrane. Further studies with similar models would aid better understanding of corneal diseases.

Purpose: To investigate the toxic effect of air on primary human retinal pigment epithelium cells (RPE) over time., Methods: RPE cultures were retrieved from six donor eyes and cultivated at 37 °C in 5% CO(2) and 95% humidified air. The RPE were divided in six groups with each group containing four samples. Six groups of RPE cultures were set up and four samples were enclosed in each group: group 1 consisted of samples in which RPE were exposed to air for 1 hour. Group 2 consisted of RPE exposed to air for 3 hours, group 3 for 6 hours group 4 for 12 hours group 5 for 24 hours and group 6 for 36 hours respectively., Results: Six hours after exposure, the morphology of the cells was still intact. At 12 hours few cells appeared enlarged. 24 hours after exposure to air the cells started losing their typical morphology and appeared deformed. Viability was higher than 95% in groups 1-3 while for groups 4-6 was 75.5%, 15.5% and 7.3%, respectively., Conclusion: The toxic effect of air for the studied in vitro model of RPE is not significant for the first 6 hours. The morphology of the cells progressively changed after 12 hours of exposure and almost all cells appeared apoptotic at 36 hours.

Purpose: To investigate the possible toxic effect of air exposure for an in vitro model of primary human ocular trabecular meshwork cells (HTM)., Methods: HTM were isolated from five donor eyes and cultivated at 37 °C. After reaching confluence the cells were seeded on two well chamber slides. The chamber slides were turned upside down in a Petri culture dish full of culture medium and filled with air using a 5 ml syringe, starting this way the exposure of the cells to the air. Subsequently they were placed in the incubation chamber at 37 °C. Six groups of HTM cultures were set up: group 1 consisted of samples in which HTM were exposed to air for 30 min, group 2 for 1 h, group 3 for 3 h, group 4 for 6 h, group 5 for 12 h and group 6 for 24 h., Results: At 3 h after exposure, the morphology of the cells was still intact, at 6 h few cells appeared deformed and exhibited characteristics of more senescent cells. At 12 h after exposure to air the HTM cells started losing their typical morphology and appeared enlarged and compromised. Viability was superior to 94% in groups 1-3 while for groups 4, 5, 6 it was 82.7%, 39.5% and 12.7% respectively., Conclusion: The toxic effect of air exposure for the studied in vitro model of HTM is not significant for the time period of one to three hours. However it starts reducing viability and alternating morphology 6 h after exposure until the time period of 24 h, where the percentage of living cells is drastically decreased. Therefore, we suggest that the use of an air bubble especially in glaucomatous patients should be applied with caution., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: To determine the time-dependent toxicity of Trypan blue at 0.06% concentration in cultured human trabecular meshwork cells., Methods: Human trabecular meshwork cells cultured in vitro were exposed to Trypan blue and acute toxicity was evaluated. Cells were exposed for 5, 30, 60 s and for 5, 15, 30, 60 min to a commercially available Trypan blue preparation (Vision Blue; DORC International Zuidland, The Netherlands). Morphology was observed by phase-contrast microscopy and cell viability was measured using Hoechst 33342 (Intergen; Purchase, NY, USA) and propidium iodide assays to determine the percentage of living and dead cells., Results: Morphological changes occurred mainly after 5 min of exposure to Trypan blue. Viability was 96.0% ± 3.6%, 94.8% ± 3.5% and 92.5% ± 4.4% after 5, 30 and 60 s of exposure, respectively; a significant toxic effect of Trypan blue was observed after 5, 15, 30 and 60 min of exposure with a viability of 85.0% ± 3.7%, 77.0% ± 6.7%, 70.8% ± 5.9% and 68.3% ± 8.7%, respectively., Conclusions: Trypan blue is not toxic, in terms of cell viability, over an exposure time of up to 60 s; however, further exposure results in a gradual increase in damage of cultured human trabecular meshwork cells., (© 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: Collagen cross-linking using UV-A irradiation combined with the photosensitizer riboflavin is a new technique for treating progressive keratoconus. The purposes of this study were to examine whether primary human corneal keratocytes (HCKs) are capable of expressing and secreting fibronectin and tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix protein, and to examine whether fibronectin and tTgase are increased after the treatment of HCK cells with UV-A irradiation combined with riboflavin (RFUV-A), thus providing another possible physiological mechanism of the cross-linking pathway., Methods: Cell cultures established from HCKs were treated with 0.025% riboflavin solution and UV-A (370 nm) irradiance 3 mW/cm2 for 30 minutes. Induction of fibronectin and tTgase was investigated by immunohistochemistry, real-time polymerase chain reaction, and Western blot analysis. Cell viability was quantified by a microscopic live-dead assay. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin., Results: Treatment of cultured HCK cells with RFUV-A increased the fibronectin and tTgase messenger RNA and protein levels. This effect was not observed in cells treated with riboflavin or UV-A radiation alone. Incorporation of biotinylated cadaverine was significantly increased when HCK cells were treated with RFUV-A., Conclusions: The enzymes tTgase and fibronectin are expressed by RFUV-A treatment in cultured HCK cells. This mechanism provides more information about the physiology of corneal cross-linking.

Purpose: To investigate the possible toxic effect of air exposure for an in vitro model of primary human corneal endothelial cells (HCECs)., Methods: Primary HCECs were isolated from donor corneal rings and cultivated at 37°C in 5% CO2 and 95% humidified air. Six groups of HCEC cultures were set up, and 4 samples were enclosed in each group: group 1 consisted of samples in which HCECs were exposed to air for 30 minutes. Group 2 consisted of HCECs exposed to air for 1 hour, group 3 for 3 hours, group 4 for 6 hours, group 5 for 12 hours, and group 6 for 24 hours., Results: Three hours after exposure, the morphology of the cells was still intact; however, a few cells within the monolayer appeared enlarged and exhibited characteristics of more senescent cells. Six hours after exposure to air, the endothelial cells started losing their typical hexagonal morphology and appeared enlarged and compromised. Viability was superior to 95% in groups 1 to 3, whereas for groups 4, 5, and 6 was 71%, 22.4%, and 6.3%, respectively., Conclusion: The present study illustrates that the toxic effect of air exposure for the studied in vitro model of primary human-cultured corneal endothelial cells is not significant for the period of 3 hours, whereas after 6 hours it starts to induce major apoptotic mechanisms, leading to reduced viability until the period of 24 hours where the percentage of living cells is drastically decreased.

Purpose: Alkylphosphocholines (APCs) are investigated for their effect on Müller glial cell proliferation and F-actin stress fiber distribution in vitro., Materials and Methods: Müller cells were incubated with APCs (C18:1-PC and C22:1-PC) +/- fetal calf serum. Proliferation was assessed by BrdU labeling and with the tetrazolium dye-reduction assay. Toxicity was measured using the trypan blue exclusion assay. The distribution of F-actin stress fibers was determined using FITC-phalloidin staining., Results: APCs are effective inhibitors of human and rat Müller glial cell proliferation and hypoxia-induced up-regulation of F-actin stress fibers in vitro in non-toxic concentrations., Conclusions: APCs might prevent intraretinal changes as a result of serum stimulation and hypoxia following retinal detachment.

Purpose: To investigate the cut quality and surface characteristics of the epithelial flap and underlying Bowman's membrane created by the Amadeus II (AMO) microkeratome on human corneas using light and electron microscopy., Setting: Center for Refractive Therapy, Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany., Methods: Using a 9.0 mm type II suction ring and settings, as recommended by the manufacturer, epithelial laser in situ keratomileusis (epi-LASIK) was performed in 2 fresh human eyes of 1 donor. Ocular pathology and previous ocular surgery were ruled out. Tissues for light microscopy were examined using hematoxylin-eosin and periodic acid-Schiff reaction staining. Further tissue samples were examined using scanning electron microscopy and transmission electron microscopy., Results: Light microscopy showed a thoroughly separated epithelial sheet with no evident anatomical abnormalities. Stratification of the separated epithelium layer and cell shape was conserved. The cleavage plane was located at Bowman's membrane. Scanning electron microscopy showed a consistent transition from adherent epithelium to the denuded area. Bowman's layer showed a very smooth surface without remains of basal lamina. Transmission electron microscopy examination showed interruptions of the basement membrane at high magnification., Conclusions: This in vitro study found a high cut quality using the epi-LASIK separator of the Amadeus II microkeratome. The resulting cleavage plane at Bowman's membrane was well suited for the subsequent laser ablation.

Background: To perform lamellar keratolimbal allograft transplantation in a one-step procedure with a single graft, we investigated the feasibility of harvesting eccentric lamellar keratolimbal grafts from conventionally processed corneoscleral buttons using a manually guided microkeratome in conjunction with an artificial anterior chamber system., Methods: We used the Moria LSK-One microkeratome and the automated lamellar therapeutic keratoplasty (ALTK) system (Antony, France). Ten human donor eyes were used to obtain single-piece lamellar keratolimbal grafts. Specimens were processed for light and electron microscopy., Results: Eccentric keratolimbal grafts could be obtained from all human donor buttons. Grafts include a crescent-shaped limbal and a large corneal portion. No visible damage to the limbal region was discernible., Conclusion: Our data show that the LSK-One microkeratome in conjunction with the ALTK system allows harvesting eccentric keratolimbal grafts from donor corneoscleral buttons.

Purpose: Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease., Methods: Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-beta2, basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-beta2-modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-beta2 treatment., Results: mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-beta2 treatment. Silencing of CTGF expression did not attenuate the TGF-beta2-mediated induction of kTgase., Conclusions: The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-beta2. TGF-beta2-mediated increase seems to be independent of CTGF.

Purpose: To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery., Methods: Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (ARPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%., Results: All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro., Conclusions: The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

Purpose: To investigate transglutaminases (enzymes capable of cross-linking extracellular matrix proteins to proteolysis-resistant complexes during scar tissue formation) in a human donor cornea after successful laser in situ keratomileusis (LASIK) without clinical complications and to compare with the results in a human donor cornea with corneal scarring after corneal injury., Setting: Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany., Methods: A donor cornea with prior uneventful LASIK treatment and 1 with corneal scarring after penetrating injury were investigated. Cryostat sections were stained immunohistochemically for tissue transglutaminase (tTG), keratocyte transglutaminase (kTG), and their reaction product epsilon-(gamma-glutamyl)-lysine., Results: With light microscopy, the flap interface of the LASIK-treated eye could hardly be detected, while in the injured eye, infiltration of cells and a clear margin next to the scar formation were present. Immunohistochemistry demonstrated a distinct staining for tTG, kTG, and epsilon-(gamma-glutamyl)-lysine in the corneal scar. In contrast, neither transglutaminase nor epsilon-(gamma-glutamyl)-lysine staining could be observed at the flap margin or in the interface of the LASIK-treated donor eye., Conclusions: Irreversible protein cross-linking of transglutaminases via epsilon-(gamma-glutamyl)-lysine connections seem to be indicators for scarring in corneal wound healing. The absence of transglutaminases and their reaction product epsilon-(gamma-glutamyl)-lysine in a LASIK-treated cornea supports the idea of missing scar tissue formation after LASIK surgery.

Background: The purpose of this study was to evaluate the influence of previous argon laser trabeculoplasty (ALT) on transforming growth factor-beta 2 (TGF-beta 2) concentration of the aqueous humor and its influence on bleb scarring after trabeculectomy., Methods: Fifty-one patients with primary open-angle glaucoma (POAG) and 29 patients with exfoliation (XFS) glaucoma were recruited for this prospective study before undergoing trabeculectomy. Sixty to 200 micro l of aqueous humor were analyzed for total and biologically active TGF-beta 2 concentrations (R and D Systems). TGF-beta 2 levels and a standardized bleb assessment were compared between the ALT- and non-ALT-treated groups., Results: POAG eyes without ALT showed significantly higher total TGF-beta 2 levels (2,317.7+/-1,041.1 pg/ml) than eyes with previous ALT (1,621.6+/-899.6 pg/ml; P=0.026). No significant difference was found for active TGF-beta 2 levels (ALT: 238.1+/-119.0 pg/ml; no ALT: 220.1+/-96.9 pg/ml; P=0.585). In XFS patients ALT did not alter total TGF-beta 2 levels (ALT: 1,524.9+/-624.9 pg/ml, no ALT: 1,220+/-499.1 pg/ml; P=0.20), but active TGF-beta 2 was significantly higher in the ALT-treated (237.0+/-99.7 pg/ml) than in the non-ALT-treated (140.0+/-95.3 pg/ml, P=0.028) group. Bleb grading revealed no statistical difference between the ALT- and non-ALT-treated groups in POAG (P=0.545, Fisher's exact test), whereas XFS patients with ALT were at increased risk for scarring compared to non-ALT-treated patients (P=0.053)., Conclusions: ALT appears to increase the risk of scarring in XFS patients because of increased levels of activated TGF-beta 2.

Purpose: In 2001, more than one million laser in situ keratomileusis (LASIK) procedures were performed worldwide. Considering the increasing number of refractive procedures, eye banks will be increasingly confronted with the problem of how to identify those donors with prior refractive surgery. To date, efficient screening methods to identify LASIK surgery in donor eyes have not been established. Therefore, the purpose of the current study was to determine whether optical coherence tomography (OCT) can be used to detect the presence of LASIK-induced changes in human corneas., Methods: Laser in situ keratomileusis was performed on 20 organ-cultured human cornea disks. The excimer laser ablation performed ranged from 0 to 12 diopters. The corneas were maintained in culture, and the visibility of flap-stromal interface by OCT was assessed up to 6 months after the LASIK procedure. Additionally, two donor corneas with the history of LASIK treatment before death were screened for structural changes., Results: Optical coherence tomography scans were able to detect the interface between the corneal flap and the residual stromal tissue in all corneas and at all examined time intervals. There were no differences in signal intensity among the different depths of ablation. The relative signal intensity of the interface compared with the averaged stromal intensity ranged from 2.1 to 6.0. In both donor corneas with suspected prior LASIK surgery, OCT scanning showed the characteristic stromal interface as found in the in vitro model., Conclusions: Corneal examination by OCT could be an appropriate technique for eye banks to screen donor corneas for prior LASIK surgery.

Purpose: Recurrent or persistent corneal erosions and ulcerations are typical complications of atopic keratoconjunctivitis. Toxic eosinophil granule proteins such as major basic protein (MBP) and eosinophil cationic protein (ECP) may be involved in this pathogenetic process. This study was designed to demonstrate the presence of toxic eosinophil granule proteins in corneal tissue from a patient with corneal complications of atopic keratoconjunctivitis., Design: Observational case report., Methods: Three corneal buttons of a patient with atopic keratoconjunctivitis associated ulcerations or scarring were examined by light microscopy and by immunofluorescence technique., Results: A linear deposition of eosinophil granular substance was detected subepithelially above Bowman's membrane in all corneal buttons. Indirect immunofluorescence identified this material as MBP and ECP. The deposits were not limited to the area of ulceration, but were also found underneath intact corneal epithelium. Multiple eosinophils were present in the upper corneal stroma. Normal corneas and negative control sections of the pathologic buttons revealed only minimal nonspecific staining at the surface of the epithelium., Conclusions: Both MBP and ECP are known to affect human corneal epithelial cell viability and morphology in vitro. Moreover, MBP was shown to inhibit epithelial migration and protein synthesis. These toxic eosinophil proteins may also be responsible for corneal instability, recurrent and persistent corneal epithelial defects and ulcerations in patients with atopic keratoconjunctivitis.

Purpose: To determine the cause of unilateral dilated tortuous episcleral vessels in a 34-year-old patient., Methods: The patient underwent slit-lamp examination, visual field testing, tonographic measurement, orbital ultrasound examination, orbital color Doppler blood flow measurement, dye-enhanced computed topographic scan, and selective carotid angiography., Results: Disk cupping and early scotoma were present. Gonioscopy showed Schlemm's canal engorged with blood. The only pathologic findings were an increased tonographic resistance to outflow and increased arterial and venous episcleral flow on color-coded Doppler sonography., Conclusion: The origin of this episcleral vessel abnormality is still unknown.