Comparison between the techniques of inclusion in glycol methacrylate (GMA)- based plastic resin and paraffin for evaluation intestinal morphometry in horses
PDF

Keywords

biopsy, equine, intestinal mucosa, small intestine, villi height

How to Cite

Ferreira, C., Palhares, M. S., de Melo, U. P., Chiarini-Garcia, H., Maranhão, R. de P. A., Gheller, V. A., & Leme, F. O. P. (2022). Comparison between the techniques of inclusion in glycol methacrylate (GMA)- based plastic resin and paraffin for evaluation intestinal morphometry in horses. Brazilian Journal of Veterinary Medicine, 44, e004521. https://doi.org/10.29374/2527-2179.bjvm004521

Abstract

To determine the effect of the inclusion method on the histomorphometric evaluation of the gastrointestinal mucosa of horses, jejunum samples were collected using flank laparotomy. Sixteen mixed breed healthy adult horses, including four males and 12 females, aged 4–14 years with an average body weight of 248.40 ± 2.28 kg, were used. Jejunal biopsies were collected and analyzed by light microscopy using two methods: group 1 comprised biopsies fixed using 10% neutral formalin and embedded in paraffin; biopsies in group 2 were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, followed by inclusion in glycol methacrylate (GMA)-based plastic resin. Intestinal villi height, crypt depth, glandular mucosa thickness, total mucosal thickness, and villus/crypt ratio were then evaluated. For all the variables studied, with exception of the villus/crypt ratio, a significant difference (p < 0.05) was found between samples in groups 1 and 2. Processing samples for embedding in plastic resin was quicker and easier to perform compared to that for paraffin embedding. In addition, the epithelial lining of tissues in group 2 showed better resolution for conducting cytological studies under a light microscope. The difference between the studied variables can be attributed to tissue retraction caused by conventional processing for inclusion in paraffin. Therefore, the method of inclusion in GMA described in the present study appears to be a more reliable choice for morphometric evaluation of the intestinal mucosa of horses.

https://doi.org/10.29374/2527-2179.bjvm004521
PDF

References

Amaral, D., Chiarini-Garcia, H., Vale Filho, V. R., & Allen, W. R. (2004). Efeito dos fixadores formalina e Bouin na preservação de biópsias de endométrio de éguas após inclusão em resina plástica. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 56(1), 7-12. http://dx.doi.org/10.1590/S0102-09352004000100002.

Batista, F. A., Zucoloto, S., Alessi, A. C., Souza, A. I., Nogueira, E., & Valadão, C. A. A. (2005). Histomorfometria dos neurônios do plexo mioentérico do cólon descendente de equinos expostos ao cloreto de benzalcônio. Ars Veterinária, 21, 193-198.

Bezerra, D. O., Carvalho, M. A. M., Conde Júnior, A. M. C., Vieira, A. A. R., Sousa, R. P., Monteiro, H. M. A., Silva, M. L. G., Soares, L. L. S., & Neves, C. A. (2016). Morphological study of cecum from agouti (Dasyprocta prymnolopha) bred in captivity. Brazilian Journal of Veterinary Medicine, 38(2), 181-186.

Bozzola, J. J., & Russell, L. D. (1998). Electron microscopy: Principles and techniques for biologists (2nd ed.). Boston: Jones & Bartlett Publishers.

Campos, L. S., Tafuri, W. L., & Pinto, A. J. W. (2016). Avaliação de diferentes fixadores na preservação das características histológicas de pele de orelha de cães. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 68(5), 1212-1218. http://dx.doi.org/10.1590/1678-4162-8595.

Carvalho, K. S. (2009). Influência do formol utilizado para conservação de cadáveres na obtenção de DNA nuclear em tecido muscular [Dissertação de mestrado em Biologia Bucal]. Universidade Estadual de Campinas, Piracicaba.

Chiarini-Garcia, H., Parreira, G. G., & Almeida, F. R. (2011). Glycol Methacrylate embedding for improved morphological, morphometrical, and immunohistochemical investigations under light microscopy: Tests as a model. In H. Chiarini-Garcia & R. Melo (Eds.), Light Microscopy: Methods in Molecular Biology (Methods and Protocols), 689. Totowa: Humana Press. http://dx.doi.org/10.1007/978-1-60761-950-5_1.

Faleiros, R. R., Macoris, D. G., Alves, G. E. S., Saquetti, C. H. C., & Alessi, A. C. (2007). Avaliação histomorfométrica e ultraestrutural da mucosa do cólon menor equino submetido à distensão. Pesquisa Veterinária Brasileira, 27(9), 383-387. http://dx.doi.org/10.1590/S0100-736X2007000900005.

Flax, M. H., & Caulfield, J. B. (1962). Use of methacrylate embedding in light microscopy. Archives of Pathology, 74, 387-395. PMid:13945443.

Junqueira, L. C., & Carneiro, J. (1999). Histologia básica (9. ed.). Rio de Janeiro: Guanabara Koogan.

Kalck, K. A. (2009). Inflammatory bowel disease in horses. The Veterinary Clinics of North America. Equine Practice, 25(2), 303-315. http://dx.doi.org/10.1016/j.cveq.2009.04.008. PMid:19580941.

Kirkeby, S., & Moe, D. (1986). Studies on the actions of glutaraldehyde, formaldehyde, and mixtures of glutaraldehyde and formaldehyde on tissue proteins. Acta Histochemica, 79(1), 115-121. http://dx.doi.org/10.1016/ S0065-1281(86)80108-8. PMid:3090833.

Lima, D. K. S., Pessoa, M. S., Arhnold, E., Leite, P. R. S. C., Leonídio, A. R. A., Santos, R. L. R., Eickstein, C., Pinto, V. M., & Abrão, F. O. (2020). Intestinal and immunological histological parameters of broilers supplemented with commercial probiotic or fungi of the autochthonous microbiota. Brazilian Journal of Veterinary Medicine, 42, e101220. http://dx.doi.org/10.29374/2527-2179.bjvm101220.

Lucas, F. A., Cattelan, J. W., Duarte, C. A., Boleli, I. C., & Malheiros, E. B. (2005). Aspectos morfométricos da mucosa do cólon descendente de equinos. Ars Veterinária, 21(2), 215-219.

Luna, L. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (Chap. 4, pp. 32-46). New York: McGraw-Hill Book. Routine staining procedures.

Melo, U. P., Palhares, M. S., Ferreira, C., França, S. A., Serakides, R., & Silva Filho, J. M. (2014). Efeito de diferentes soluções de enema sobre as características macro e microscópicas da mucosa do cólon menor e reto de equinos. A Hora Veterinária, 34(1), 49-52.

Mendes, H. M. F., Escobar, A., Vasconcelos, A. C., Zucoloto, S., Alves, G. E. S., & Faleiros, R. R. (2009). Alterações morfométricas no plexo mioentérico do cólon menor equino distendido experimentalmente. Pesquisa Veterinária Brasileira, 29(7), 557-562. http://dx.doi.org/10.1590/S0100-736X2009000700010.

Milne, E. M., Pirie, R. S., McGorum, B. C., & Shaw, D. J. (2010). Evaluation of formalin-fixed ileum as the optimum method to diagnose equine dysautonomia (Grass Sickness) in simulated intestinal biopsies. Journal of Veterinary Diagnostic Investigation, 22(2), 248-252. http://dx.doi.org/10.1177/104063871002200214. PMid:20224086.

Rocchigiani, G., Ricci, E., Navarro, M. A., Samol, M. A., & Uzal, F. A. (2021). Leukocyte numbers and intestinal mucosal morphometrics in horses with no clinical intestinal disease. Journal of Veterinary Diagnostic Investigation, 10406387211031944. http://dx.doi.org/10.1177/10406387211031944. PMid:34293980.

Ruemmele, F. M., Schmitz, J., & Goulet, O. (2006). Microvillous inclusion disease (microvillous atrophy). Orphanet Journal of Rare Diseases, 1(1), 22. http://dx.doi.org/10.1186/1750-1172-1-22. PMid:16800870.

Tacha, D. E., & Richards, T. C. (1981). Comparison of glycol methacrylate and paraffin-embedded tissues for identification of Tritium-labeled cells after emulsion radioautography. Journal of Histotechnology, 4(2), 59-62. http://dx.doi.org/10.1179/his.1981.4.2.59.

SAS USER’S guide: statistics. Release 8.1 Cary, NC: SAS Institute Inc. 2000.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Copyright (c) 2022 Cíntia Ferreira, Maristela Silveira Palhares, Ubiratan Pereira de Melo, Helio Chiarini-Garcia, Renata de Pino Albuquerque Maranhão, Valentim Arabicano Gheller, Fabíola Oliveira Paes Leme