Morphology of Endothelial Cells from Different Regions of the Swine Cornea

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-month-old, 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 evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea

Morphology of endothelial cells from different regions of the swine cornea

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 evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea

Morfologia das células do endotélio de diferentes regiões da córnea de suínos (sus scrofa domesticus) utilizando vermelho de alizarina e microscopia óptica

O endotélio da córnea realiza a função importante na manutenção da transparência da mesma e regulação da hidratação do estroma. É composto por uma monocamada de células poligonais, achatadas e interligadas que recobrem a superfície posterior da córnea. Dentre as técnicas utilizadas para avaliação do endotélio, o vermelho de alizarina é uma técnica in vitro de baixo custo, de rápida e fácil execução. Objetivou-se avaliar as células do endotélio da córnea de suínos adultos em suas diferentes regiões, utilizando a coloração por vermelho de alizarina e microscopia óptica. Para esse estudo, foram utilizados 24 bulbos oculares de 12 suínos abatidos, 120 dias de idade, da raça Large White, adultos, machos ou fêmeas. As córneas foram removidas, e o endotélio corado com vermelho de alizarina dissolvido previamente em solução isotônica (0,2 g/100 ml), com pH ajustado para 4,2 com ácido clorídrico e avaliadas no microscópio óptico. Foram feitas fotografias das regiões central, inferior, lateral, medial e superior do endotélio da córnea e, após, foi realizado o estudo do formato de 100 células. Para análise estatística, foi utilizado o teste de variância (ANOVA por medidas repetidas). A percentagem média de células hexagonais na região central do globo ocular direito foi de 82,75%± 6,355, na região inferior 84,83% ± 4,108, na lateral 86,0%± 4,090, na região medial 83,50%± 2,447 e na região superior 83,17%± 4,345. No globo ocular esquerdo, a percentagem média de células hexagonais na região central foi de 81,92 % ±2,539, na região inferior foi de 83,67% ± 3,339, na região lateral de 84,00% ±3,618, na região medial 84,17%± 3,010 e na região superior 82,83% ± 4,064. Não houve diferença estatística significativa com relação à morfologia entre as diferentes regiões da córnea e entre os globos oculares direito e esquerdo. A coloração com vermelho de alizarina e a microscopia óptica foram eficazes para avaliação do formato das células do endotélio da córnea de suínos. Os valores obtidos, com relação ao formato das células da região central da córnea, representam todo o mosaico endotelial, podendo ser extrapolados para as regiões periféricas.The corneal endothelium perform are important function in maintaing corneal transparency and regulating stromal hydration. It consists of monolayer polygonal cells, fattened and joined, covering of the posterior surface of the cornea. Among the technique used for evaluating the endothelium, Alizarin red vital dye is a in vitro technique for low coast, fast and easy perform. This study aimed to evaluated the endothelial cells from adult pigs cornea in its different regions used the coloration by Alizarin red and, optical microscopy. For this study we used 24 eye bulbs of 12 slaughtered pigs, 120 day of age, of Large White breed, adults, male or female. The cornea was removed and the endothelium stained with Alizarin red, previously dissolved in isotonic solution (0,2g/100 ml), pH adjusted to 4,2 with hydrochloric acid, and evaluated in optical microscope. Photographs were taken of the central regions, lateral, medial and upper corneal endothelium. After the study was performed of 100 cells format. For statistical analysis we used the analysis of variance (ANOVA for repeated measures). The average percentage of hexagonal cells in the central region in the right eye was 82,75%±6,355, in the lower 84,83% ± 4,108, on lateral 86,0%± 4,090, in the medial region 83,50%± 2,447 and the upper region 83,17%± 4,345. In the left eye, the percentage of hexagonal cells in the central region was 81,92 % ±2,539, in lower 83,67% ± 3,339, in lateral 84,00% ±3,618, in medial 84,17%± 3,010 and in upper region 82,83%± 4,064. There was no statistically significant difference in the relation morphology between the different regions of the cornea and between the right and left eyeballs. Staining with Alizarin red and optical microscopy were effective for evaluating the shape of the endothelial cells of the pig cornea. The values obtained in relation of the shape of the central cornea cells, represent the entire endothelial mosaic and can be extrapolated to the peripheral regions

Morphology of Endothelial Cells from Different Regions of the Swine Cornea

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 &amp; Results: Twenty-four healthy eyes from 12 swine Large White breed, with 14-month-old, 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 &lt; 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 evaluating the shape of the endothelial cells in the five different regions of the cornea. This study has demonstrated that the parameters evaluated in swine did not differ significantly between the various places of the cornea