Sheep corneal endothelium morphology - evaluation with trypan blue and alizarin red

Background: The endothelium is a layer fundamental to maintaining corneal transparency. In ophthalmology, sheep eyes have been used as a model in research related to corneal transplantation. Different techniques have been used to evaluate the corneal endothelium. Concerning vital dyes, corneal endothelial cell analyses have not yet been studied in ovines. The purpose of the present study was to evaluate the morphology of endothelial cells from different regions of the cornea of sheep after staining with alizarin red and trypan blue using an optical microscope. Materials, Methods & Results: Twenty healthy eyes of 10 male sheep obtained from a licensed commercial slaughterhouse were studied. The study was approved by the Research Committee of the Faculty of Veterinary at UFRGS and followed the ethical standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after the slaughter, the eyes were enucleated and underwent eye examination. The corneal endothelium was stained with trypan blue and alizarin red and examined and photographed using an optical microscope. The central, superior, inferior, nasal and temporal areas of the cornea were evaluated for cell morphology. Data were compared by t-tests. Differences were considered statistically significant at P < 0.05. Immediately after staining the corneal endothelium, it was possible to examine with an optical microscope, obtain images and analyse the shape of endothelial cells from all regions of the sheep cornea. Polygonal, uniform and continuous cells were observed in all samples studied. Considering all the corneas analysed, cells with 6 sides (75.11%), 5 sides (12.76%) and 4 sides (12.12%) were found. In the central region of the cornea 75.91% of cells with 6 sides, 12.6% of cells with 5 sides and 11.48% with 7 sides were found. In the superior region of the cornea 76.07% of cells with 6 sides, 13.25% with 5 sides and 10.68% with 7 sides were found. In the lower region were found 74.72% of cells with 6 sides, 13% with 5 sides and 12.27% with 7 sides. In the temporal region, 74.14% were 6-sided cells, 11.42% had 5 sides, and 14.43% had 7 sides. Furthermore, in the nasal region, 74.72% of the cells had 6 sides, 13.54% had 5 sides, and 11.73% had 7 sides. No significant differences were found between cell morphology in all corneal regions evaluated. In addition, no significant difference was found when comparing the right eye with the left eye. Discussion: Different methods are used for the analysis of corneal endothelium. For ex vivo research optical microscopy after endothelial staining is an alternative low-cost technique that allows the analysis of all regions of the cornea. Quantitative analyses must characterise the endothelial parameters of the different species. 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 with the borated cells, allowing identification. In the present study, using optical microscopy and coloration with alizarin red it was possible to explore and obtain images of the ovine endothelium of all regions of the cornea. In the current study, the endothelium had a predominance of cells will 6 sides in all regions studied. This study allowed us to obtain images of the endothelium as well as quantitative data on the morphology of the different regions of the sheep cornea. This study demonstrated that morphology did not differ between the central and peripheral regions. The findings of this study represent a further source of reproducible data that should be considered when using sheep cornea as ex vivo model for experimental research.

Corneal endothelial cell density and morphology in rabbits eyes using contact specular microscopy / Densidade celular e morfologia das células do endotélio da córnea de coelhos obtidas com microscopia especular

The goal of this study was to describe the normal values of corneal endothelial cell density and hexagonality in healthy rabbits eyes using contact specular microscopy. Both eyes of 18 rabbits, males and females, of different ages were evaluated. The eyes were divided into three groups of 12 each according to the age range of the animals: G1 (6 months old), G2 (12 months old), and G3 (48 months old). Parameters studied included mean endothelial cell density and hexagonality. For Group I, the mean cell density was 2307 cells per mm2. For GII, the mean cell density was 1895 cells per mm2. For G III, the mean cell density was 1818 cells per mm2. Cell density decreased significantly with aging (P 0.001) among the three groups. Pleomorphism in the rabbits of GI was 74.33±10.08. In the rabbits of GII it was 71.83±11.38. In the rabbits of GIII it was 64.02±28.80. Significant differences (P 0.001) were evident between GI and GIII and GII and GIII but not between Groups I and II. The results showed a decrease in number of cells and hexagonality with aging. Corneal endothelium of rabbits suffers changes due to advancing age both in terms of cell density and morphology.

Objetivou-se avaliar a densidade celular e a hexagonalidade do endotélio da cَrnea de coelhos saudلveis usando um microscَpio especular de contato. Os dois olhos de 18 coelhos, machos ou fêmeas, de diferentes idades foram avaliados. Os olhos foram separados em três grupos de 12 olhos cada em funçمo da idade: G1 (6 meses de idade), G2 (12 meses de idade) e G3 (48 meses de idade). Os parâmetros estudados incluيram a densidade celular e a hexagonalidade. Para o Grupo I, a densidade celular média foi de 2307 células por mm2. Para GII, a densidade celular média foi de 1895 células por mm2. Para G III, a densidade celular média foi de 1818 células por mm2. A densidade celular diminuiu significativamente com o envelhecimento (P 0,001) entre os três grupos. O pleomorfismo nos coelhos de GI foi de 74,33±10,08. Nos coelhos do GII foi 71,83±11,38. Nos coelhos de GIII foi de 64,02±28,80. Diferenças significativas (P 0,001) foram evidentes entre GI e GIII e GII e GIII, mas nمo entre os Grupos I e II. Os resultados revelaram diminuiçمo da densidade celular e da hexagonalidade com o avanço da idade. O endotélio da cَrnea de coelhos sofre alteraçُes devido ao avanço da idade tanto na densidade celular e quanto na morfologia.

Corneal endothelial cell density and morphology in rabbits eyes using contact specular microscopy / Densidade celular e morfologia das células do endotélio da córnea de coelhos obtidas com microscopia especular

The goal of this study was to describe the normal values of corneal endothelial cell density and hexagonality in healthy rabbits eyes using contact specular microscopy. Both eyes of 18 rabbits, males and females, of different ages were evaluated. The eyes were divided into three groups of 12 each according to the age range of the animals: G1 (6 months old), G2 (12 months old), and G3 (48 months old). Parameters studied included mean endothelial cell density and hexagonality. For Group I, the mean cell density was 2307 cells per mm2. For GII, the mean cell density was 1895 cells per mm2. For G III, the mean cell density was 1818 cells per mm2. Cell density decreased significantly with aging (P 0.001) among the three groups. Pleomorphism in the rabbits of GI was 74.33±10.08. In the rabbits of GII it was 71.83±11.38. In the rabbits of GIII it was 64.02±28.80. Significant differences (P 0.001) were evident between GI and GIII and GII and GIII but not between Groups I and II. The results showed a decrease in number of cells and hexagonality with aging. Corneal endothelium of rabbits suffers changes due to advancing age both in terms of cell density and morphology.(AU)

Objetivou-se avaliar a densidade celular e a hexagonalidade do endotélio da cَrnea de coelhos saudلveis usando um microscَpio especular de contato. Os dois olhos de 18 coelhos, machos ou fêmeas, de diferentes idades foram avaliados. Os olhos foram separados em três grupos de 12 olhos cada em funçمo da idade: G1 (6 meses de idade), G2 (12 meses de idade) e G3 (48 meses de idade). Os parâmetros estudados incluيram a densidade celular e a hexagonalidade. Para o Grupo I, a densidade celular média foi de 2307 células por mm2. Para GII, a densidade celular média foi de 1895 células por mm2. Para G III, a densidade celular média foi de 1818 células por mm2. A densidade celular diminuiu significativamente com o envelhecimento (P 0,001) entre os três grupos. O pleomorfismo nos coelhos de GI foi de 74,33±10,08. Nos coelhos do GII foi 71,83±11,38. Nos coelhos de GIII foi de 64,02±28,80. Diferenças significativas (P 0,001) foram evidentes entre GI e GIII e GII e GIII, mas nمo entre os Grupos I e II. Os resultados revelaram diminuiçمo da densidade celular e da hexagonalidade com o avanço da idade. O endotélio da cَrnea de coelhos sofre alteraçُes devido ao avanço da idade tanto na densidade celular e quanto na morfologia.(AU)