The study has been conducted on 40 species of mammals included in different taxonomic orders: 10 carnivores (felines and canides), 13 primates and 17 herbivores (artiodactyles, perisodactyles and proboscideans). Domestic, experimental and wild animals have been included in the sample. The purpose of the study is to offer new data regarding morphometric aspects of the epithelium of the cornea of mammals. We also aim to establish the relationships and differences between the different epithelia studied and to determine the morphometric characteristics which best define the epithelium of the different species under study. The results obtained have demonstrated the existence of correlations between thicknesses, number of cellular layers and number of the epithelial cells in the different groups. It has also been established that the variants in thickness and number of layers define and characterize the epithelia. Finally, the study reveals that morphometric characteristics of the epithelium of primates and carnivores have more similarities than those of herbivores.

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 ≥ 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.

The aim of the present paper is to describe the morphological changes that occur in human corneal endothelium as an immediate consequence of corneal cryopreservation. Therefore, 16 human donor corneas were cryopreserved with an original procedure at a 1 °C/min cooling rate in a freezing solution cryoprotected with 7% dimethylsulphoxide until a final temperature of –100 °C was reached. After storage of the corneas in liquid nitrogen for periods ranging from 1 to 96 days (mean: 34.31 days), the corneas were thawed in a water bath at +37 °C. Eight additional control corneas were processed without cryopreservation. Morphological assessment of the endothelial layer was performed by scanning electron microscopy and trypan blue and alizarin red S vital staining. Results showed cryoinduced damage at variable degrees in all cryopreserved corneas. They were classified into three groups according to the intensity and extension of the cryoinduced damage: group I (n = 10): corneas with minor endothelial alterations consisting in the presence of microholes in the posterior cell membrane; group II (n = 1): corneas with generalized disruption of endothelial intercellular junctions and intact cell membranes; group III (n = 5): corneas with severe endothelial damage consisting of massive cell necrosis and complete alteration of the morphological pattern of the endothelium. All control corneas had intact endothelial layers. Cryoinduced damage cannot be completely avoided with the cryopreservation protocol tested. The high interindividual variability of the results observed is not related to the storage time of the cornea in liquid nitrogen.

Morphological and morphometric features of the cornea of 13 species of primates have been studied in order to determine possible morphological differences between them. The existence of relationships between different morphometric corneal variables was also examined to establish which variables best defined and characterized the cornea. The present aim is to determine which primate cornea resembles that of the human being most with a view to possible future clinical and experimental studies. The results obtained revealed that all the cornea under study presented similar morphological features. The relationship between total corneal thickness and corneal epithelial thickness was determined as well as the relationship between epithelial thickness, the number of epithelial layers and the number of epithelial cells. However, the morphological pattern of Bowman's membrane and corneal endothelium differed in the species studied. Finally, the study indicates that the chimpanzee and the gorilla are the species with a corneal morphometry which is closest to that of the human cornea.

The purpose of the present study was to develop an optimal freezing method for cryopreservation of human donor corneas for transplantation. Three groups of ten human donor corneas each were cryopreserved using cooling rates of 0.5 degree C/ min, 1 degree C/min and 9 degrees C/min. The freezing medium contained 10% fetal calf serum and 7% dimethylsulphoxide. Ten additional human donor corneas were used as controls. Endothelial cell survival after complete thawing was assessed by calculating the mean endothelial cell density and percentage of non-viable endothelial cells from vital staining and scanning electron micrographs. Significant differences in endothelial cell survival between all groups were detected by analysis of variance (p < 0.001), but paired contrast found no real differences between corneas frozen at 0.5 degree C/min or 1 degree C/min. Nevertheless, 60% of the corneas cryopreserved at 1 degree C/min but only 10% of those cryopreserved at 0.5 degree C/min fulfilled both requirements to be considered suitable for transplantation (endothelial cell density > 2000 cells/mm2 and less than 10% dead cells). None of the corneas frozen at 9 degrees C/min fulfilled these requirements. Mean endothelial cell density of corneas frozen at 1 degree C/min was 2084 cells/mm2 (range 2020 to 2630 cells/mm2). Except for isolated corneas frozen at 0.5 degree C/min, only corneas cryopreserved at a cooling rate of 1 degree C/min achieved satisfactory endothelial cell survival for their use in transplantation. However the significant interindividual variability among corneas frozen at 1 degree C/min (ANOVA: p < 0.001) prevents prediction of the condition of the cornea after the complete cryopreservation process. Until this high variability can be drastically reduced, systematic corneal cryopreservation in eye banking remains unattainable.