Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae.

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (-183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.

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/mm(2). 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/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) 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/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.

The application of autologous serum eye drops in severe dry eye patients; subjective and objective parameters before and after treatment.

AIM: To assess the impact of autologous serum (AS) eye drops on the ocular surface of patients with bilateral severe dry eye and to draw a comparison between the clinical and laboratory examinations and the degree of subjective symptoms before and after serum treatment. MATERIALS AND METHODS: A three-month prospective study was conducted on 17 patients with severe dry eye. AS eye drops were applied a maximum of 12 times a day together with regular therapy. Dry eye status was evaluated by clinical examination (visual acuity, Schirmer test, tear film breakup time, vital staining, tear film debris and meniscus), conjunctival impression cytology (epithelial and goblet cell density, snake-like chromatin, HLA-DR-positive and apoptotic cells) and subjectively by the patients. RESULTS: The application of AS eye drops led to a significant improvement in the Schirmer test (p < 0.01) and tear film debris (p < 0.05). The densities of goblet (p < 0.0001) and epithelial cells (p < 0.05) were significantly increased, indicating a decrease of squamous metaplasia after AS treatment. A significant decrease (p < 0.05) was found in the number of apoptotic, HLA-DR-positive and snake-like chromatin cells on the ocular surface. A significant improvement was found in all evaluated subjective symptoms. Altogether, the clinical results were improved in 77%, the laboratory results in 75% and the subjective feelings in 63% of the eyes. CONCLUSIONS: We found that three-month AS treatment led especially to the improvement of ocular surface dryness and damage of the epithelium. The improvement of dry eye after AS treatment correlated well with the clinical, laboratory and subjective findings. From the patients' subjective point of view, the positive effect of AS decreased with time, but still persisted up to three months after the end of therapy.

Endothelial cell density before and after the preparation of corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim.

PURPOSE: To assess the endothelial cell density before and after the preparation of precut posterior corneal lamellae for Descemet membrane (DM) endothelial keratoplasty with a stromal rim with the aim of standardizing this new technique and establishing the parameters of corneas intended for manual preparation under the conditions of an ocular tissue bank. METHODS: Two groups of corneoscleral buttons were used to prepare lamellae consisting of a central zone of endothelium-DM surrounded by a supporting peripheral stromal rim. Group 1 contained 12 corneas with a live endothelial cell density (LECD) of 2500 cells per square millimeter or more. Group 2 contained 10 corneas with a density of 2500 cells per square millimeter or less. The preparation was performed manually using an artificial anterior chamber. The endothelial cell density (ECD) and the percentage of dead cells were assessed before and immediately after preparation and after 48 hours of organ culture storage at 31°C. RESULTS: Immediately after preparation, on average, 4.9% and 5.2% of dead cells were found in group 1 and group 2, respectively. After 48 hours of storage, the percentage decreased significantly in both the groups. There was no significant decrease in the ECD 48 hours after preparation in group 1; however, there was a significant decrease in group 2. The amount of tissue wasted during preparation was 24%. CONCLUSIONS: The decrease in ECD is dependent on the initial values: the cell loss is less in corneas with higher original densities. We suggest that the minimal acceptable LECD of lamellae should be 2500 cells per square millimeter.

The assessment of pathogenic prions in the brains of eye tissue donors: 2-years experience in the Czech Republic.

PURPOSE: The aim of this study was to assess the presence of pathogenic prions in the brain tissue of eye donors and to evaluate the benefits of 2-year obligatory testing in the Czech Republic. METHODS: Brain tissue was retrieved during autopsies of eye donors of 3 tissue banks in the Czech Republic. The frozen specimens obtained from the frontal lobe were transported to the Czech National Reference Laboratory for the diagnosis of human prion disorders. The presence of pathogenic prions was tested using the Prionics-Check WESTERN kit. Confirmative Western blotting using 1 of 2 different clones of monoclonal anti-PrP antibody was performed as well. RESULTS: No pathogenic prions were found in any of the 1142 tested specimens. One specimen revealed weak positivity at initial screening; however, repeated examination of the specimen and other specimens from different locations in the brain of the same donor did not confirm the presence of pathogenic prions. The negative result was confirmed by the National CJD Surveillance Unit, University of Edinburgh, United Kingdom. CONCLUSION: The absence of pathogenic prions from all of the 1142 tested specimens corresponds to the presumed very low risk of transmission of Creutzfeldt-Jakob disease through corneal graft transplantation. As a result of this disorder's rarity, a larger series of tested samples should be evaluated to obtain statistically significant findings. Although such testing increases the safety of donor eye tissue, it also increases the expense, causes organizational difficulties, and may extend the time needed to release the tissue for grafting.