Acta Scientiae Veterinariae
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Morphology of Endothelial Cells from Different Regions of the Swine Cornea
Lídia Luzia Clerot, Paula Stieven Hünning, Marcelle Bettio, Marcela Torikachvili, Michelle Beck Petersen, Alessandra Fernandez Silva, Andre Silva Carissimi, João Antonio Tadeu Pigatto
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.
Abib F.C., Holzchuh R., Schaefer A., Schaefer T. & Godois R. 2012. The endothelial sample size analysis in corneal specular microscopy clinical examinations. Cornea. 31(5): 546-550.
Albuquerque L., Freitas L.V.R. & Pigatto J.A.T. 2015. Analysis of the corneal endothelium in eyes of chickens using contact specular microscopy. Semina: Ciências Agrárias. 36(6): 4199-4206.
Bercht B.S., Albuquerque L., Araujo A.C. & Pigatto J.A.T. 2015. Specular microscopy to determine corneal endothelial cell morphology and morphometry in chinchilas (Chinchilla lanigera) in vivo. Veterinary Ophthalmology. 18(1): 137-142.
Coyo N., Penã M.T., Costa D., Ríos J., Lacerda R. & Leiva M. 2015. Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Veterinary Ophthalmology. 19(5): 367-372.
Faganello C.S., Silva V.R.M., Andrade M.C.C., Carissimi A.S. & Pigatto J.A.T. 2016. Morphology of endothelial cells from different regions of the equine cornea. Ciência Rural. 46(12): 2223-2228.
Franzen A.A., Pigatto J.A.T., Abib F.C., Albuquerque L. & Laus J.L. 2010. Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes. Veterinary Ophthalmology. 13(4): 222-226.
Geroski D.H. & Edelhauser H.F. 1989. Morphometric analysis of the corneal endothelium. Specular microscopy vs. alizarin red staining. Investigative Ophthalmology & Visual Science. 30(2): 254-259.
Hara H. & Cooper D.K.C. 2011. Xenotransplantation - the future of corneal transplantation? Cornea. 30(4): 371-378.
Lee S.E., Mehra R., Fujita M., Roh D.S., Long C., Lee W., Funderburgh J.L., Ayares D.L., Cooper D.K. & Hara H. 2014. Characterization of porcine corneal endothelium for xenotransplantation. Seminars Ophthalmology. 29(3): 127-135.
Park S., Fong A.G., Cho H., Zhang C., Gritz D.C., Mian G., Herzlich A.A., Gore P., Morganti A. & Chuck R.S. 2012. Protocol for vital dye staining of corneal endothelial cells. Cornea. 31(12): 1476-1479.
Pigatto J.A.T., Andrade M.C., Laus J.L., Santos J.M., Brooks D.E., Guedes P.M. & Barros P.M.S. 2004. Morphometric analysis of the corneal endothelium of yacare caiman (Caiman yacare) using scanning electron microscopy. Veterinary Ophthalmology. 7(3): 205-208.
Pigatto J.A.T., Abib F. C., Pizzeti J.C., Laus J.L., Santos J. M. & Barros P.S.M. 2005. Análise morfométrica do endotélio corneano de coelhos à microscopia eletrônica de varredura. Acta Scientiae Veterinariae. 33(1): 41-45.
Pigatto J.A.T., Laus J.L., Santos J.M., Cerva C., Cunha L.S., Ruoppolo V. & Barros P.S.M. 2005. Corneal endothelium of the magellanic penguin (Spheniscus magellanicus) by scanning electron microscopy. Journal of Zoo and Wildlife Medicine. 36(4): 702-705.
Pigatto J.A.T., Abib F.C., Pereira G.T., Barros P.S.M., Freire C.D. & Laus J.L. 2006. Density of corneal endothelial cells in eyes of dogs using specular microscopy. Brazilian Journal of Veterinary Research and Animal Science. 43(4): 476-480.
Pigatto J.A.T., Cerva C., Freire C.D., Abib F.C., Bellini L.P., Barros P.S.M. & Laus J.L. 2008. Morphological analysis of the corneal endothelium in eyes of dogs using specular microscopy. Pesquisa Veterinária Brasileira. 28(9): 427- 430.
Pigatto J.A.T., Franzen A.A., Pereira F.Q., Almeida A.C.V.R., Laus J.L., Santos J.M., Guedes P.M. & Barros P.S.M. 2009. Scanning electron microscopy of the corneal endothelium of ostrich. Ciência Rural. 39(3): 926-929.
Proulx S. & Brunette I. 2012. Methods being developed for preparation, delivery and transplantation of a tissue-engineered corneal endothelium. Experimental Eye Research. 95(1): 68-75.
Ruggeri A., Scarpa F., Massimo L., Meltendorf C. & Schroeter J. 2009. A system for the automatic estimation of morphometric parameters of corneal endothelium in alizarine red-stained images. British Journal of Ophthalmology. 94(5): 643-647.
Saad H.A., Terry M.A., Shamie N., Chen E.S., Friend D.F., Holiman J.D. & Stoeger C. 2008. An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dying staining and adobe photoshop software. Cornea. 27(7): 818-824.
Silva V.R.M., Andrade M.C.C., Faganello C.S., Torikachvili M., Carissimi A.S. & Pigatto J.A.T. 2018. Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study. Semina: Ciências Agrárias. 39(2): 613-620.
Smeringaiova I., Merjava S.R., Stranak Z., Studeny P., Bednar J. & Jirsova K. 2018. Endothelial wound repair of the organ-cultured porcine cornea. Current Eye Research. 43(7): 856-865.
Tamayo-Arango L.J., Baraldi-Artoni S.M., Laus J.L., Mendes-Vicenti F.A., Pigatto J.A.T. & Abib F.C. 2009. Ultrastructure, morphology and morphometry of a normal corneal endothelium of adult crossbred pig. Ciência Rural. 9(1): 117-122.
Taylor M.L. & Hunt C.J. 1981. Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction. British Journal of Ophthalmology. 65(12): 815- 819.
Terzariol M., Hünning P.S., Brambatti G., Albuquerque L., Neumann C. & Pigatto J.A.T. 2016. Effects of intracameral brilliant blue on the corneal endothelium of swine: in vitro study. Pesquisa Veterinária Brasileira. 36(8): 775-780.
Copyright (c) 2019 Lídia Luzia Clerot, Paula Stieven Hünning, Marcelle Bettio, Marcela Torikachvili, Michelle Beck Petersen, Alessandra Fernandez Silva, Andre Silva Carissimi, João Antonio Tadeu Pigatto