Morphology of Endothelial Cells from Different Regions of the Swine Cornea

Abstract

Background:The corneal endothelium is a monolayer of polygonal cells which constitute the last layer of the cornea. The integrity of this layer is critical to cornea transparency. The characterization of normal corneal endothelial morphology is important not only to clinical evaluation but also to selection of areas of the cornea with better quality to be employed as donor tissue. The aim of the present study was to evaluate the morphology of endothelial cells from different regions of the swine cornea after alizarin red staining using optical microscopy. Materials, Methods & Results:Twenty-four healthy eyes from 12 swine Large White breed, with 14-monthold, males or females obtained from a licensed Brazilian commercial slaughterhouse were studied. Immediately after humane slaughter, the eyes were enucleated and submitted to ophthalmic examination. Eyes with signs of diseases of the anterior segment were excluded. The cornea, with 3 mm of the sclera, was removed and placed on a glass microscope slide with the endothelial side up. Four radial incisions were made in the periphery of the cornea to better accommodate the cornea on the microscope slide. Alizarin red was diluted in isotonic solution (0.2 g/100 mL) and the pH was adjusted to 4.2 with hydrochloric acid. Three drops of alizarin red were placed on the corneal endothelium. After 90 s, the dye was removed from the cornea with balanced saline solution. The corneal endothelium was examined and photographed using an optical microscope. All evaluations were performed by the same investigator. Photomicrographs were taken of central, superior, inferior, nasal and temporal corneal areas. Parameter studied included endothelial cell morphology. For the statistical analysis, was employed the ANOVA variance test (repeated measures). Differences were considered statistically significant at P < 0.05. Normal endothelium cells were mainly hexagonal (83.7%), pentagonal (7.45%) and heptagonal (8.8%), with a minimal number of cells of other shapes present. There were no significant statistical differences in the proportion of the morphology and the different regions of the cornea (P = 0.31). Discussion: Different techniques are available for the analysis of corneal endothelium, including mainly scanning electron microscopy, specular microscopy and optical microscopy. The analysis of the morphology of corneal endothelium with an optic microscope after staining with alizarin red has been described as an effective, rapid and cost-efficient method, since this dye blends the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and to obtain images of the swine endothelium of all regions of the cornea. The analysis of the cellular morphology or the percentage of hexagonal cells are among the main parameters used to evaluate the health of the corneal endothelium. In this study, the endothelium had the predominance of the hexagonal shape in all regions studied. In swine, there are no studies...

Microscopia especular das diferentes regiões da córnea de suínos - estudo ex vivo

“…Os suínos tem sido amplamente utilizados como modelo experimental em oftalmologia devido às semelhanças de suas córneas com a córnea humana (27,32,33,28) . No entanto, estudos avaliando o endotélio corneano saudável de suínos são escassos (28,30,31,34) , e variações interespécies nas características do endotélio corneano impedem a extrapolação dos dados obtidos de uma espécie para outra. Devido a essa variação dos parâmetros endoteliais, o ideal é estabelecer valores de referência em córneas saudáveis para cada espécie.…”

“…No presente estudo, não houve diferença significativa na porcentagem de células hexagonais entre as cinco regiões avaliadas. Da mesma forma, Clerot et al (31) , usando microscopia óptica e corantes vitais, também não encontraram diferenças significativas na hexagonalidade de diferentes regiões de uma córnea suína saudável. Os resultados obtidos no presente estudo mostram que não houve diferenças na DCE e na hexagonalidade quando os olhos direito e esquerdo foram comparados.…”

“…Devido às semelhanças entre suínos e humanos tem aumentado o interesse pela possibilidade de utilização de córneas suínas para transplante em humanos (27,28) . No entanto, estudos avaliando o endotélio corneano dessa espécie são escassos (28)(29)(30)(31) .…”

Specular microscopy of the different regions of the cornea in enucleated swine eyes - ex vivo evaluation

“…The swine has widely been used as an experimental model in ophthalmology due to similarities of its cornea to the human cornea (27,32,33,28) . However, studies evaluating the healthy corneal endothelium of pigs are scarce (28,30,31,34) , and interspecies variations in the characteristics of the corneal endothelium prevent the extrapolation of data obtained from one species to another. Due to this variation in endothelial parameters, the ideal is to establish reference values in healthy corneas for each species.…”

“…Due to the similarities between swine and human, the interest in the possibility of using swine corneas for transplantation in humans has increased (27,28) . However, studies evaluating the corneal endothelium of this species are scarce (28)(29)(30)(31) .…”