Indicators of progressive corneal exposure to dry eye conditions.

PURPOSE: To establish the main indicator of progressive corneal exposure to dry eye conditions in an animal model. METHODS: Cell surface covered by microvilli (SCM), intercellular junctions (IJs), cell area, cell shape, and cell shade were measured in 988 epithelial corneal cells from rabbit eyes that were kept open for different drying times (DT) between 0 and 3 h. Two multivariate logistic regressions were used to model the relationship between these morphometric cell characteristics and DT. To corroborate the results, terminal differentiation was assessed by cluster analysis. RESULTS: Scanning electron microscopy images of the epithelial cells showed typical desiccation changes that increased in frequency and intensity as DT were prolonged. Binomial logistic regression, to distinguish cells exposed to desiccation from the control cells, displayed SCM, IJ, and cell shade as significant indicators (p < 0.01) of desiccation exposure. Multinomial logistic regression analysis showed SCM, IJ, cell shade, and cell area as significant indicators (p < 0.01) differentiating the four levels of desiccation exposure, and SCM was the variable that showed elevated odds ratio in all the analysis. In addition, the cells were grouped in three groups (G1, G2, and G3) by cluster analysis. G2 cells increased in percentage as DT grew (p < 0.05, χ2 test) in detriment of G1 cells, while the percentage of G3 cells remained stable. These changes were consistent with a terminal differentiation process from G1 cells to G2 cells, which was the group with the lowest values of SCM (p < 0.001, Mann-Whitney test). CONCLUSIONS: The variable SCM could be the best determining indicator of progressive corneal exposure to dry eye conditions. The development of clinical methods to analyze microvilli density could allow a more accurate assessment of the level of degradation of the corneal epithelium under evaporative conditions.

Image processing techniques in a preliminary morphometric characterization of corneal epithelium by scanning electron microscopy.

Several processing techniques of digital images allowed us to quantify the percentage of cell surface covered by microprojections (microvilli or microplicae) (SCM), the adhesion between epithelial cells by the parameter intercellular junctions (IJ), the size (cell area), shape (cell shape) and shade (cell shade) of cells on the corneal epithelium of nine rabbits. The data were analyzed and the epithelial cells were classified into three groups by cluster analysis. Assuming the representativeness of the sample, our findings suggest that for a normal corneal epithelium, 80% of the cells could show SCM >41%, and IJ >0.98 (being one a cell to cell junction without disruptions). Standard deviations of cell shade lower than 21 gray levels could indicate a tendency to lose the cell shade mosaic. Normal corneas could show a majority of cells (54-69%) included in group 2 with smaller mean size (80% of cells with cell area <242 µm(2)), higher SCM (80% of cells with SCM >44.83%), polygonal mean shape and brighter shade. Group 1 (15-30% of cells) could show a larger mean size (80% of cells with cell area >494 µm(2)), lower SCM (although 80% of cells with SCM >32.61%), circular mean shape and darker electron reflex. Group 3 could display a medium mean size, higher SCM (similar to group 2), circular mean shape (similar to group 1), and brighter shade. These analyses could possibly be used to further assess the corneal response to ocular drugs, contact lens, and surgical procedures or to discriminate between pathology stages.

Image processing techniques to quantify microprojections on outer corneal epithelial cells.

It is widely accepted that cellular microprojections (microvilli and/or microplicae) of the corneal surface are essential to maintain the functionality of the tissue. To date, the characterization of these vital structures has been made by analysing scanning or transmission electron microscopy images of the cornea by methods that are intrinsically subjective and imprecise (qualitative or semiquantitative methods). In the present study, numerical data concerning three microprojection features were obtained by an automated method and analysed to establish which of them showed less variability. We propose that the most stable microprojection characteristic would be a useful sign in early detection of epithelial damage or disease. With this aim, the scanning electron microscopy images of 220 corneal epithelial cells of nine rabbits were subjected to several image processing techniques to quantify microprojection density, microprojection average size and surface covered by microprojections (SCM). We then assessed the reliability of the methods used and performed a statistical analysis of the data. Our results show that the thresholding process, the basis of all image processing techniques used in this work, is highly reliable in separating microprojections from the rest of the cell membrane. Assessment of histogram information from thresholded images is a good method to quantify SCM. Amongst the three studied variables, SCM was the most stable (with a coefficient of variation of 15.24%), as 89.09% of the sample cells had SCM values > or = 40%. We also found that the variability of SCM was mainly due to intercellular differences (the cell factor contribution represented 88.78% of the total variation in the analysed cell areas). Further studies are required to elucidate how healthy corneas maintain high SCM values.

Comparative study of ablation profiles of six different excimer lasers.

PURPOSE: To compare the morphological features of photorefractive ablations produced by six different excimer lasers. METHODS: A spherocylindrical photoablation (-2.00 -2.00 x 90 degrees; 6-mm optical zone) was performed on regular fluence plates with six excimer lasers: Bausch & Lomb Technolas 217C, Schwind Esiris, Kera Technology Isobeam D200, Ladarvision 4000, Zeiss Meditec MEL 70 G-Scan, and Visx Star S3. Morphometric analysis of the fluence plates provided superficial measurements of the ablated areas. RESULTS: Two areas were identified visually: a central area with a complete ablation of the metallic surface layer and a surrounding area with a partial ablation. The dimensions of the ablated areas were highly variable in the lasers tested. The major differences appeared in the total ablated area (ranging from 38.55 mm2 [Schwind] to 81.94 mm2 [Bausch & Lomb] and in the peripheral to total area ratio (ranging from 36.95% [Schwind] to 59.51% [Ladarvision]). CONCLUSION: Large differences appeared in the superficial dimensions and contours of the ablations produced by different excimer lasers for the treatment tested in this study. It remains unknown how these different ablation patterns induce the same optical correction, but we assume that the depth of the ablation compensates for the differences in the surface extension of the ablated areas.