Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm². The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm² and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm² and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm² in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.     

Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas

The posterior side of the cornea is covered by the endothelial monolayer, which governs corneal transparency but cannot proliferate. Determination of endothelial cell density (ECD) is therefore the minimal and mandatory quality control in all eye banks. It avoids primary graft failures caused by endothelial insufficiency, and allows allocation of corneas to surgical techniques requiring different numbers of endothelial cells (ECs). Corneas stored in organ culture (17% of grafts worldwide), are characterized by heavy stromal swelling and numerous deep endothelial folds, up to 200 µm high. During microscopic en face observation, flat surfaces are thus exceptional and EC counting is biased by parallax errors, resulting in overestimated eye bank ECD (ebECD). We used a motorized transmitted light microscope to acquire Z-stacks of images every 10 µm, and processed them to reconstruct the 3D surface of the folded endothelium. This method (3D-ECD) takes into account the local point-by-point slope in order to correct ECD. On a set of 30 corneas, we compared 3D-ECD and ebECD determined on five identical zones at the center of the cornea. 3D reconstruction allowed us to visualize twice as many cells, and ebECD was 8.1 ± 4.5% (95%CI 6.4–9.7) higher than 3D-ECD, with 1744 ± 488 versus 1606 ± 473 cells/mm². 3D counting makes it possible to increase cell sampling and to correct overestimation by the conventional en face counting still routinely performed in eye banks.     

Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks

The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm² by steps of 400 cells/mm²) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 μm² (incremental steps of 25 μm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 μm² grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 μm² grid counts. Digital image analysis with a variable frame counting method would be the best alternative.     

Cell adhesion molecules in stromal corneal dystrophies

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.     

Tectonic epikeratoplasty with ethanol-stored donor corneas

To evaluate the efficacy and outcomes of tectonic epikeratoplasty with use of ethanol-preserved corneal grafts for the management of perforated corneal melts. The present retrospective case series includes 10 eyes which underwent tectonic epikeratoplasty for perforated corneal melts. The stromal remainders of Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft preparation were stored in 95% ethanol and used as emergency tectonic grafts for restoring globe integrity after sterile and infectious perforated corneal melts. In 6 cases with subtotal corneal melt, DMEK remainders (endothelium-denuded corneoscleral buttons) were used for ‘limbus to limbus’ tectonic epikeratoplasty and in 4 cases DSAEK remainders (anterior stroma) were used to seal focal perforated melts. Graft storage time was 5.1?±?4.9 (ranging from 0.5 to 17) months. The surgeries were successful in all cases with restitution of the globe integrity. During the postoperative course 4 cases developed a graft melt (corneoscleral button for limbus to limbus tectonic epikeratoplasty, n?=?3; lamellar patch, n?=?1) within 2–6 months after the initial procedure. Three patients underwent successful repeat tectonic epikeratoplasty. In the fourth case of graft melt the globe was enucleated due to underlying expulsive haemorrhage and severe pain. The short-term results of the present case series suggest that the use of ethanol-stored stromal remainder of donor corneas after endothelial keratoplasty is an efficient temporary measure for tectonic restoration of perforated corneas.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Proinflammatory conditions promote hepatocellular carcinoma onset and progression via activation of Wnt and EGFR signaling pathways

The aim of the current study was to investigate how proinflammatory conditions affect growth and progression of hepatocellular carcinoma. Human hepatoma cell lines were treated with lipopolysaccharide (LPS) or cyclooxygenase-2 inhibitor, Celecoxib, and in vitro proliferation, apoptosis, and cell cycle progression were assessed. This was followed up with in vivo xenograft assays to monitor tumor growth and metastatic progression under different treatment conditions. While LPS induced cell proliferation, Celecoxib induced apoptosis. Flow cytometry analysis demonstrated that S-phase cell count in LPS group was higher than control group (41.9 ± 3.2 vs 30.6 ± 0.1 %, respectively), whereas G0/G1-phase cells were significantly higher in the Celecoxib group in comparison with the control group (69.6 ± 5.0 vs 50.4 ± 1.6 %, respectively) (p < 0.05). Immunoblot analyses showed induction of epidermal growth factor receptor expression and induction and nuclear accumulation of Wnt/β-catenin and p65 in LPS group. Xenograft assays showed that LPS treatment induced comparatively large, rapidly growing tumors (2,702 ± 572 mm³) that metastasized to lungs, whereas Celecoxib treatment alone (1,008 ± 296 mm³) or in combination with LPS (1,303 ± 283 mm³) suppressed tumor growth in comparison to control groups (2,072 ± 456 mm³) (n = 5; p < 0.05). Inflammation can thus promote hepatoma cell proliferation and growth, and enhance the invasion and metastatic ability of hepatocarcinoma cells through inducing tumor angiogenesis, which in turn may be related to the activation of Wnt/β-catenin and EGFR signaling pathways.     

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers.?The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec^®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at ?80°C, monolayers were rapidly thawed and re-cultured in cell culture medium.?Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of ?1°C/min.?In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.     

Angiopeptin,a somatostatin-14 analogue,decreases adhesiveness of rat Leukocytes to unstimulated and IL-1β-activated rat heart endothelial cells

We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131–140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1β-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 ± 12 leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 μM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 ± 12 to 188 ± 10, 185 ± 8 and 172 ± 3 cells/mm², respectively. Stimulation of endothelial cells with IL-1β (100U/ml) for 24 hours increased endothelial adhesiveness from 274 +- 12 to 381 ± 17 adhering leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1μM) reduced significantly (p < 0.05) binding of leukocytes to IL-1β-activated endothelium from 381 ± 17 to 237 ± 8, 254 ± 11 and 248 ± 13 cells/mm², respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CDlla/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1β-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.     

Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

A quantitative method to evaluate the donor corneal tissue quality used in a comparative study between two hypothermic preservation media

To standardize a new evaluation technique for calculating the overall quality (OQ) of the donor cornea and validate it using a comparative study of corneas preserved in Optisol-GS and Cornea Cold^®. Thirty pairs of donor corneas were selected for a 4 week in vitro comparative study using masked observers. Physiological parameters like thickness, transparency, viable endothelial cell density (VECD) and morphology were transformed to numerical range (0–4) to obtain the OQ. Microbiological examination was performed using Bactec instrument. Students t test showed statistically better results (p < 0.05) from week 3 for thickness, week 2 for transparency and week 1 for morphology and VECD; statistical significance (p < 0.05) was found for OQ from week 2 for the corneas preserved in Cornea Cold^® compared to Optisol-GS. Epithelial quality was similar regardless of the medium. Microbiological examination showed absence of aerobic and anaerobic microorganisms in both media. OQ method is efficient, consistent and easy, now validated for comparative studies. Further refinement is necessary for its use at eye-banks, bio-banks and research or transplantation purposes. Cornea Cold^® is a promising hypothermic corneal storage medium with preservation time ≤21 days. This permits higher flexibility, evaluation accuracy, longer duration for surgical preparation and ease of transportation.     

Abscopal effect of boron neutron capture therapy (BNCT): proof of principle in an experimental model of colon cancer

The aim of the present study was to evaluate, for the first time, the abscopal effect of boron neutron capture therapy (BNCT). Twenty-six BDIX rats were inoculated subcutaneously with 1 × 10⁶ DHD/K12/TRb syngeneic colon cancer cells in the right hind flank. Three weeks post-inoculation, the right leg of 12 rats bearing the tumor nodule was treated with BPA-BNCT (BPA-Boronophenylalanine) at the RA-3 nuclear reactor located in Buenos Aires, Argentina, at an absorbed dose of 7.5 Gy to skin as the dose-limiting tissue. The remaining group of 14 tumor-bearing rats were left untreated and used as control. Two weeks post-BNCT, 1 × 10⁶ DHD/K12/TRb cells were injected subcutaneously in the contralateral left hind flank of each of the 26 BDIX rats. Tumor volume in both legs was measured weekly for 7 weeks to determine response to BNCT in the right leg and to assess a potential influence of BNCT in the right leg on tumor development in the left leg. Within the BNCT group, a statistically significant reduction was observed in contralateral left tumor volume in animals whose right leg tumor responded to BNCT (post-treatment/pre-treatment tumor volume <1) versus animals who failed to respond (post/pre ≥1), i.e., 13 ± 15 vs 271 ± 128 mm³. In addition, a statistically significant reduction in contralateral left leg tumor volume was observed in BNCT-responsive animals (post/pre <1) vs untreated animals, i.e., 13 ± 15 vs 254 ± 251 mm³. The present study performed in a simple animal model provides proof of principle that the positive response of a tumor to BNCT is capable of inducing an abscopal effect.     

Hypercalcemia induces a proinflammatory phenotype in rat leukocytes and endothelial cells

Endothelial plasma membrane lipid microdomains, so called lipid rafts/caveolae, are rich in neutral glycosphingolipid, globotriaosylceramide, Gb3Cer, or CD77. Several plasma membrane Ca²⁺ channels and pumps are located in lipid rafts/caveolae. Increased Ca²⁺ influx could cause the development of an endothelial proinflammatory phenotype. Therefore, the aim of this study was to estimate the effects of hypercalcemia in rats by determination of CD77 expression on CD34+ endothelial cells in the heart, kidney, and vena cava. In addition, potential proinflammatory calcium effect was estimated by CD11b and CD15s expression on leukocytes. To achieve hypercalcemia, Sprague–Dawley male rats were given CaCl₂ solution with a concentration of 1.5 % elemental calcium during 14 days. CD77 expression on CD34+ endothelial cells in cell suspensions of the heart, kidney, and vena cava, as well as leukocyte expression of CD11b and CD15s in hypercalcemic and control rats were determined by flow cytometry. Ionized calcium concentration in plasma was 1.37?±?0.01 mM in hypercalcemic vs. 1.19?±?0.03 mM in control rats. Hypercalcemic group showed statistically significantly decreased proportion of endothelial CD34+ CD77? cells in the kidney and vena cava in parallel with increase of CD11b and CD15s leukocyte proinflammatory markers. In conclusion, it is tempting to speculate that plasma membranes of glycosphingolipid CD77? endothelial cells are poorer in caveolae lipid microdomains and therefore weaker in controlling of Ca²⁺ influx. The percentage of CD11b+ CD15s+ leukocytes could be a measure of proinflammatory effects of mild hypercalcemia.     

Validation of an endothelial roll preparation for Descemet Membrane Endothelial Keratoplasty by a cornea bank using “no touch” dissection technique

Descemet Membrane Endothelial Keratoplasty (DMEK) selectively replaces the damaged posterior part of the cornea. However, the DMEK technique relies on a manually-performed dissection that is time-consuming, requires training and presents a potential risk of endothelial graft damages leading to surgery postponement when performed by surgeons in the operative room. To validate precut corneal tissue preparation for DMEK provided by a cornea bank in order to supply a quality and security precut endothelial tissue. The protocol was a technology transfer from the Netherlands Institute for Innovative Ocular Surgery (NIIOS) to Lyon Cornea Bank, after formation in NIIOS to the DMEK “no touch” dissection technique. The technique has been validated in selected conditions (materials, microscope) and after a learning curve, cornea bank technicians prepared endothelial tissue for DMEK. Endothelial cells densities (ECD) were evaluated before and after preparation, after storage and transport to the surgery room. Microbiological and histological controls have been done. Twenty corneas were manually dissected; 18 without tears. Nineteen endothelial grafts formed a double roll. The ECD loss after cutting was 3.3 % (n = 19). After transportation 7 days later, we found an ECD loss of 25 % (n = 12). Three days after cutting and transportation, we found 2.1 % of ECD loss (n = 7). Histology found an endothelial cells monolayer lying on Descemet membrane. The mean thickness was 12 ± 2.2 µm (n = 4). No microbial contamination was found (n = 19). Endothelial roll stability has been validated at 3 days in our cornea bank. Cornea bank technicians trained can deliver to surgeons an ECD controlled, safety and ready to use endothelial tissue, for DMEK by “no touch” technique, allowing time saving, quality and security for surgeons.     

Preparation of pre-cut corneas from fresh donated whole globes for Descemet’s stripping automated keratoplasty: 3-year results at the Central Eye Bank of Iran

To describe the technique and the results of the preparation of pre-cut corneas for Descemet’s stripping automated endothelial keratoplasty (DSAEK) during a 3-year period at the Central Eye Bank of Iran (CEBI). The method of preparation of pre-cut corneas from donated whole globes at the CEBI is described and the frequency and percentage of pre-cut corneas prepared for DSAEK, between April 2009 and March 2012, are specified. Moreover, post-operative reports are reviewed for any complaints about using pre-cut tissues for DSAEK. Out of the 1,518 donated whole globes appropriate for DSAEK, 1,478 (97.4 %) pre-cut corneas were successfully prepared. The method of preparation failed in 40 (2.6 %) cases. Based on the eye bank post-operative reports, thickness of pre-cut tissues for DSAEK was deemed unacceptable in only 6 (0.4 %) cases prior to surgery; five of these were too thick and one was too thin. Preparation of pre-cut corneas, for DSAEK from donated whole globes, in the CEBI is a safe and easy method, with very good preservation of endothelial cells after the preparation of the pre-cut corneas and reduced risks from corneal manipulation.     

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium con- taining chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul- fate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to con- fluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical re- searches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.     

Antifungal Activity of Nanocapsule Suspensions Containing Tea Tree Oil on the Growth of Trichophyton rubrum

The aim of this study was to evaluate, for the first time, the antifungal efficacy of nanocapsules and nanoemulsions containing Melaleuca alternifolia essential oil (tea tree oil) in an onychomycosis model. The antifungal activity of nanostructured formulations was evaluated against Trichophyton rubrum in two different in vitro models of dermatophyte nail infection. First, nail powder was infected with T. rubrum in a 96-well plate and then treated with the formulations. After 7 and 14 days, cell viability was verified. The plate counts for the samples were 2.37, 1.45 and 1.0 log CFU mL^?1 (emulsion, nanoemulsion containing tea tree oil and nanocapsules containing tea tree oil, respectively). A second model employed nails fragments which were infected with the microorganism and treated with the formulations. The diameter of fungal colony was measured. The areas obtained were 2.88 ± 2.08 mm², 14.59 ± 2.01 mm², 40.98 ± 2.76 mm² and 38.72 ± 1.22 mm² for the nanocapsules containing tea tree oil, nanoemulsion containing tea tree oil, emulsion and untreated nail, respectively. Nail infection models demonstrated the ability of the formulations to reduce T. rubrum growth, with the inclusion of oil in nanocapsules being most efficient.     

P53, CD95, cathepsin and survivin pathways in Fuchs' dystrophy and pseudophakic bullous keratopathy corneas

Our purpose was to elucidate the pathways of apoptosis of corneas with Fuchs' dystrophy and pseudophakic bullous keratopathy. Sixteen corneal buttons (14 patients, median age 73 years) with Fuchs' dystrophy, 13 with pseudophakic bullous keratopathy (PBK) (13 patients, median age 69 years) and 8 buttons (8 patients, median age 59 years) from enucleated eyes with chorioideal melanoma (controls) were analysed histologically. Immunohistochemical analysis was performed to investigate the expression of p21, p27, p63, survivin, CD95, cathepsin, bax, bcl-2 and Ki67. Positive immunohistochemical reactions were detected in epithelial cells of the corneas, but keratocytes and endothelial cells were not positive in any of the groups or stainings. The number of p27 and survivin positive epithelial cells was significantly lower (p=0.048 and 0.041) and the number of cathepsin positive epithelial cells was significantly higher (p=0.004) in Fuchs' dystrophy corneas compared to controls. In pseudophakic bullous keratopathy, p21 and p27 positive epithelial cells were present in a significantly lower (p=0.02 and 0.005) number than in controls. We conclude that genetically programmed cell death is related to the p27, cathepsin and survivin pathways in Fuchs' dystrophy and to the p21 and p27 pathways in pseudophakic bullous keratopathy.     

Effect of freezing and thawing on the biomechanical characteristics of porcine ocular tissues

PurposeTo evaluate the effect of freezing and thawing on the biomechanical properties of ex-vivo porcine ocular tissue.MethodsThirty-six porcine eyes (18 pairs) were obtained fresh from a local abattoir and split into two groups of nine pairs to study the effect of storage at −20 °C and −80 °C. A randomly-selected eye from each pair (Control Group, CG) was tested fresh while the fellow eyes were frozen for 14 days, either at −20 °C and −80 °C (Frozen Group, FG) before thawing and testing. Seventy-two strips were extracted from the corneas and scleras of eye globes and subjected to uniaxial tension tests under loads up to 1.0 N. Following five preconditioning cycles, the load and elongation data obtained experimentally were analysed to derive the tissue’s stress-strain and tangent modulus-strain behaviour.ResultsCorneal tissue subjected to freezing at −20 °C exhibited significant increases in tangent modulus (mechanical stiffness) by 13 ± 17% (p = 0.003) at 1% strain and 14 ± 12% (p < 0.001) at 2% strain. In contrast, the increases in corneal stiffness at −80 °C were insignificant (6 ± 14%, p = 0.099 at 1% strain, 6 ± 15%, p = 0.091 at 2% strain). The corresponding increases in tangent modulus in the sclera were all insignificant (for −20 °C: 4 ± 14%, p = 0.265 at 1% strain, 3 ± 9%, p = 0.186 at 2% strain; for −80 °C: 3 ± 18%, p = 0.537 at 1% strain and 3 ± 18%, p = 0.491 at 2% strain).ConclusionsThe study provided evidence that freezing and thawing led to insignificant changes in ocular tissue stiffness except in corneal tissue that was frozen at −20 °C.