بررسی مقایسه ای هیستومورفومتری بافت روده باریک با سه روش کلارک-بسته، فرمالین-باز و فرمالین-بسته در جوجههای گوشتی
محورهای موضوعی :
آسیب شناسی درمانگاهی دامپزشکی
عارف محمودتبار
1
(گروه پرورش و مدیریت طیور، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران)
محمد امیر کریمی ترشیزی
2
(گروه پرورش و مدیریت طیور- دانشکده کشاورزی دانشگاه تربیت مدرس- تهران- ایران)
تاریخ دریافت : 1396/10/15
تاریخ پذیرش : 1397/09/14
تاریخ انتشار : 1397/10/01
کلید واژه:
فرمالین,
روده باریک,
محلول کلارک,
هیستومرفومتری,
پایدار‌سازی بافت,
چکیده مقاله :
پایدار نمودن بافت یک مرحله بسیار حیاتی در آماده سازی بافت جهت مطالعات بافت شناسی به شمار می رود و هرگونه اشتباه در این مرحله غیرقابل جبران خواهد بود. هدف از این مطالعه مقایسه تاثیر سه روش تهیه مقاطع بافتی کلارک-بسته، فرمالین-باز و فرمالین-بسته بر هیستومورفومتری مخاط روده باریک در جوجه های گوشتی بود. در این بررسی 30 قطعه جوجه گوشتی سویهراس 308 نر در سن 10 روزگی مورد استفاده قرار گرفت. نمونه های روده باریک بعد از شستشو، بلافاصله با محلول سالین- فرمالین 10 درصد به‌شکل بسته و یا بازشده و محلول کلارک به‌صورت دو‌طرف بسته پایدار شدند. پس از پایداری طبق روش استاندارد بافت‌شناسی، مقاطع میکروسکوپی به ضخامت 5 میکرومتر تهیه و با ائوزین-هماتوکسیلین و آلسین‌بلو رنگ‌آمیزی شدند. در هر مقطع ارتفاع پرز، ضخامت پرز، عمق کریپت و تعداد سلول های جامی با خط‌کش چشمی اندازه‌گیری و شاخص سطح پرز نیز محاسبه گردید. داده ها در قالب طرح کاملاً تصادفی با آزمون دانکن مورد مقایسه قرار گرفتند. نتایج نشان داد که روش تهیه و پایداری نمونه‌های روده باریک بر شاخص های کمی بافت شناسی تاثیر معنی داری داشت (01/0>p). روش فرمالین-بسته بلند ترین ارتفاع پرز و بیشترین عمق کریپت و روش کلارک-بسته، کمترین مقادیر را نشان دادند (01/0>p). همچنین روش کلارک-بسته و روش های فرمالین-بسته و باز به ترتیب بیشترین ضخامت پرز را در هر سه قسمت روده کوچک نشان دادند (01/0>p) و بیشترین مقدار برای سطح پرز نیز در روش فرمالین-بسته مشاهده شد (01/0>p). با توجه به سادگی روش کار، قیمت مناسب، حفظ بهتر مخاط روده و عدم ایجاد تغییرات شدید در بافت، روش فرمالین-بسته به عنوان روش برگزیده معرفی می شود.
چکیده انگلیسی:
Tissue fixation is a vital step in preparation of samples for histological studies and mistakes at this stage could lead to irreversible damage. The aim of this study was to compare the effect of three sample preparation-fixation methods on histomorphometic indices of small intestine of broilers. Thirty 10 day old broilers of Ross 308 strain were used in the study. Segments of small intestine were washed and prepared as opened or closed sections. The closed sections were fixed in 10% saline-formalin. The opened samples were cut at the mesenteric line and were fixed by 10% saline-formalin. In Clarke’s fixation the ends of samples were sealed by string and Clarke’s solution was injected into the lumen. Fixed samples were subjected to paraffin embedding and 5 µm sections were cut using rotary microtome and sections were stained by Eosin-Hematoxylin and Alcian blue. In each section the villus height, villus thickness, crypt depth and goblets’ density were measured under light microscope and the villus surface index was also calculated. Data were subjected to ANOVA in a completely randomized design with three treatments and 30 observations. The results indicated that method of preparation-fixation had a significant effect on morphometric indices (p<0.01). The closed-formalin’s samples had the greatest villus height and crypt depth values in all three intestinal segments, while the lowest values were observed in closed-Clarke’s method (p<0.01). Closed-Clarke’s samples had the thickest villus in all three parts of small intestine followed by closed-formalin’s and opened-formalin’s samples (p<0.01). Villus surface area was highest in closed-formalin fixed samples (p<0.01). Considering simplicity of the method, low cost, better mucosal protection and lack of severe tissue changes observed in closed-formalin’s method, this method was considered as a method of choice in histomorphometric studies of poultry small intestinal mucosa.
منابع و مأخذ:
Aviagen (2014). ROSS 308 Broiler Nutrition Specifications. http://en.aviagen.com.
Belloni, B., Lambertini, C., Nuciforo, P., Phillips, J., Bruening, E., Wong, S., et al. (2013). Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system. Journal of Clinical Pathology, 66(2): 124-135.
Boom, A., Daems, W.T. and Luft, J.H. (1974). On the fixation of intestinal absorptive cells. Journal of Ultrastructure Research, 48(3): 350-360.
Buesa, R.J. (2008). Histology without formalin? Annals of Diagnostic Pathology, 12(6): 387-396.
Cabrera, N.C., Espinoza, J.R., Vargas-Jentzsch, P., Sandoval, P., Ramos, L.A. and Aponte, P.M. (2017). Alcohol-based solutions for bovine testicular tissue fixation. Journal of Veterinary Diagnostic Investigation, 29(1): 91-99.
Carson, F.L. and Martin, J.H. (1979). Formaldehyde as a fixative for light and electron microscopy. Journal of Histotechnology, 2(2): 58-60.
Choi, H.R., Kim, S.K., Kwon, S.B. and Park, K.C. (2006). The fixation of living skin equivalents. Applied Immunohistochemistry and Molecular Morphology, 14(1): 122-125.
Choi, J.H., Bennett, A.E. and McDonald, L.G. (2011). Effects of fixation on gastrointestinal biopsies. Journal of Histotechnology, 34(1): 40-43.
Cummins, A.G., Steele, T.W., LaBrooy, J.T. and Shearman, D.J. (1988). Maturation of the rat small intestine at weaning: changes in epithelial cell kinetics, bacterial flora, and mucosal immune activity. Gut, 29(12): 1672-1679.
Fox, C.H., Johnson, F.B., Whiting, J. and Roller, P.P. (1985). Formaldehyde fixation. Journal of Histochemistry and Cytochemistry, 33(8): 845-853.
Gava, M.S., Moraes, L.B., Carvalho, D., Chitolina, G.Z., Fallavena, L.C.B., Moraes, H.L.S., et al. (2015). Determining the best sectioning method and intestinal segment for morphometric analysis in broilers. Brazilian Journal of Poultry Science, 17(2): 145-150.
Gill, G.W. (2012). Cytopreparation: principles and practice. Springer Science and Business Media. pp: 101-129.
Groelz, D., Sobin, L., Branton, P., Compton, C., Wyrich, R. and Rainen, L. (2013). Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Experimental and Molecular Pathology, 94(1): 188-194.
Holda, M.K., Klimek‐Piotrowska, W., Koziej, M., Piątek, K. and Holda, J. (2016). Influence of different fixation protocols on the preservation and dimensions of cardiac tissue. Journal of Anatomy, 229(2): 334-340.
Hopwood, D. (1985). Cell and tissue fixation, 1972-1982. The Histochemical Journal, 17(4): 389-442.
Jones, M.L. (2013). To fix, to harden, to preserve–fixation: a brief history. Journal of Histotechnology, 24(3): 155-162.
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Kik, M.J.L., Huisman, J., van Der Poel, A.F.B. and Mouwen, J.M.V.M. (1990). Pathologic changes of the small intestinal mucosa of pigs after feeding Phaseolus vulgaris beans. Veterinary Pathology Online, 27(5): 329-334.
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Sakamoto, K., Hirose, H., Onizuka, A., Hayashi, M., Futamura, N., Kawamura, Y., et al. (2000). Quantitative study of changes in intestinal morphology and mucus gel on total parenteral nutrition in rats. Journal of Surgical Research, 94(2): 99-106.
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SAS Institute. (1990). SAS/STAT User's guide. Version 6.03. SAS institute Inc., Cary, NC. pp: 126-178.
Sasani, F. (2004). Necropsy procedures and sampling. 1st ed., Iran: University of Tehran, pp: 146. [In Persian]
Singhal, P., Singh, N.N., Sreedhar, G., Banerjee, S., Batra, M. and Garg, A. (2016). Evaluation of histomorphometric changes in tissue architecture in relation to alteration in fixation protocol–An in vitro study. Journal of Clinical and Diagnostic Research, 10(8): 28-32.
Soltan, M.A. (2009). Influence of dietary glutamine supplementation on growth performance, small intestinal morphology, immune response and some blood parameters of broiler chickens. International Journal of Poultry Science, 8(1): 60-68.
Speilberg, L., Evensen, O., Bratberg, B. and Skjerve, E. (1993). Evaluation of five different immersion fixatives for light microscopic studies of liver tissue in Atlantic salmon Salmo salar. Diseases of Aquatic Organisms, 17(1): 47-55.
Sugiharto, S. (2016). Role of nutraceuticals in gut health and growth performance of poultry. Journal of the Saudi Society of Agricultural Sciences, 15(2): 99-111.
Teshfam, M., Saeidi, J. and Zarei, A. (2010). Morphological and enzymological studies of the small intestine villi of rats receiving diets containing different levels of protein. Journal of Applied Animal Research, 37(2): 207-211.
Thavarajah, R., Mudimbaimannar, V.K., Elizabeth, J., Rao, U.K. and Ranganathan, K. (2012). Chemical and physical basics of routine formaldehyde fixation. Journal of Oral and Maxillofacial Pathology, 16(3): 400-405.
Tu, L., Yu, L. and Zhang, H. (2011). Morphology of rat testis preserved in three different fixatives. Journal of Huazhong University of Science and Technology [Medical Sciences], 31(2): 178-180.
Van Leeuwen, P., Mouwen, J.M.V.M., Van Der Klis, J.D. and Verstegen, M.W.A. (2004). Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance. British Poultry Science, 45(1): 41-48.
Williams, J.M., Duckworth, C.A., Vowell, K., Burkitt, M.D. and Pritchard, D.M. (2016). Intestinal preparation techniques for histological analysis in the mouse. Current Protocols in Mouse Biology, 6(2): 148-168.
Yegani, M. and Korver, D.R. (2008). Factors affecting intestinal health in poultry. Poultry Science, 87(10): 2052-2063.
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Aviagen (2014). ROSS 308 Broiler Nutrition Specifications. http://en.aviagen.com.
Belloni, B., Lambertini, C., Nuciforo, P., Phillips, J., Bruening, E., Wong, S., et al. (2013). Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system. Journal of Clinical Pathology, 66(2): 124-135.
Boom, A., Daems, W.T. and Luft, J.H. (1974). On the fixation of intestinal absorptive cells. Journal of Ultrastructure Research, 48(3): 350-360.
Buesa, R.J. (2008). Histology without formalin? Annals of Diagnostic Pathology, 12(6): 387-396.
Cabrera, N.C., Espinoza, J.R., Vargas-Jentzsch, P., Sandoval, P., Ramos, L.A. and Aponte, P.M. (2017). Alcohol-based solutions for bovine testicular tissue fixation. Journal of Veterinary Diagnostic Investigation, 29(1): 91-99.
Carson, F.L. and Martin, J.H. (1979). Formaldehyde as a fixative for light and electron microscopy. Journal of Histotechnology, 2(2): 58-60.
Choi, H.R., Kim, S.K., Kwon, S.B. and Park, K.C. (2006). The fixation of living skin equivalents. Applied Immunohistochemistry and Molecular Morphology, 14(1): 122-125.
Choi, J.H., Bennett, A.E. and McDonald, L.G. (2011). Effects of fixation on gastrointestinal biopsies. Journal of Histotechnology, 34(1): 40-43.
Cummins, A.G., Steele, T.W., LaBrooy, J.T. and Shearman, D.J. (1988). Maturation of the rat small intestine at weaning: changes in epithelial cell kinetics, bacterial flora, and mucosal immune activity. Gut, 29(12): 1672-1679.
Fox, C.H., Johnson, F.B., Whiting, J. and Roller, P.P. (1985). Formaldehyde fixation. Journal of Histochemistry and Cytochemistry, 33(8): 845-853.
Gava, M.S., Moraes, L.B., Carvalho, D., Chitolina, G.Z., Fallavena, L.C.B., Moraes, H.L.S., et al. (2015). Determining the best sectioning method and intestinal segment for morphometric analysis in broilers. Brazilian Journal of Poultry Science, 17(2): 145-150.
Gill, G.W. (2012). Cytopreparation: principles and practice. Springer Science and Business Media. pp: 101-129.
Groelz, D., Sobin, L., Branton, P., Compton, C., Wyrich, R. and Rainen, L. (2013). Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Experimental and Molecular Pathology, 94(1): 188-194.
Holda, M.K., Klimek‐Piotrowska, W., Koziej, M., Piątek, K. and Holda, J. (2016). Influence of different fixation protocols on the preservation and dimensions of cardiac tissue. Journal of Anatomy, 229(2): 334-340.
Hopwood, D. (1985). Cell and tissue fixation, 1972-1982. The Histochemical Journal, 17(4): 389-442.
Jones, M.L. (2013). To fix, to harden, to preserve–fixation: a brief history. Journal of Histotechnology, 24(3): 155-162.
Kiernan, J.A. (2001). Histological and histochemical methods (Theory and practical). Translated by: Mahdavi Shahri, N., Fazel, A., Jian Tabasi, M. and Saadatfar, Z. 2nd ed., Iran: Ferdowsi University of Mashhad, pp: 40-73. [In Persian]
Kik, M.J.L., Huisman, J., van Der Poel, A.F.B. and Mouwen, J.M.V.M. (1990). Pathologic changes of the small intestinal mucosa of pigs after feeding Phaseolus vulgaris beans. Veterinary Pathology Online, 27(5): 329-334.
Perry, C., Chung, J.Y., Ylaya, K., Choi, C.H., Simpson, A., Matsumoto, K.T., et al. (2016). A buffered alcohol-based fixative for histomorphologic and molecular applications. Journal of Histochemistry and Cytochemistry, 64(7): 425-440.
Pousty, I. and Adibmoradi, M. (2006). Histotechnique. 1st ed., Iran: University of Tehran, pp: 58&254. [In Persian]
Rezaei, M., Karimi Torshizi, M.A. and Rouzbehan, Y. (2011). The influence of different levels of micronized insoluble fiber on broiler performance and litter moisture. Poultry Science, 90(9): 2008-2012.
Rezaian, M., Sodagar Amiri, A., Akbari., M.E. and Ebrahimpoor, F. (2007). Modification of fixation in preparing avian tissues. Pajouhesh and Sazandegi, 20(1): 2-8. [In Persian]
Rinttila, T. and Apajalahti, J. (2013). Intestinal microbiota and metabolites–implications for broiler chicken health and performance. The Journal of Applied Poultry Research, 22(3): 647-658.
Sakamoto, K., Hirose, H., Onizuka, A., Hayashi, M., Futamura, N., Kawamura, Y., et al. (2000). Quantitative study of changes in intestinal morphology and mucus gel on total parenteral nutrition in rats. Journal of Surgical Research, 94(2): 99-106.
Salehnia, M. (2001) General and advance histological technique. 1st ed., Iran: Paygan, pp: 35. [In Persian]
SAS Institute. (1990). SAS/STAT User's guide. Version 6.03. SAS institute Inc., Cary, NC. pp: 126-178.
Sasani, F. (2004). Necropsy procedures and sampling. 1st ed., Iran: University of Tehran, pp: 146. [In Persian]
Singhal, P., Singh, N.N., Sreedhar, G., Banerjee, S., Batra, M. and Garg, A. (2016). Evaluation of histomorphometric changes in tissue architecture in relation to alteration in fixation protocol–An in vitro study. Journal of Clinical and Diagnostic Research, 10(8): 28-32.
Soltan, M.A. (2009). Influence of dietary glutamine supplementation on growth performance, small intestinal morphology, immune response and some blood parameters of broiler chickens. International Journal of Poultry Science, 8(1): 60-68.
Speilberg, L., Evensen, O., Bratberg, B. and Skjerve, E. (1993). Evaluation of five different immersion fixatives for light microscopic studies of liver tissue in Atlantic salmon Salmo salar. Diseases of Aquatic Organisms, 17(1): 47-55.
Sugiharto, S. (2016). Role of nutraceuticals in gut health and growth performance of poultry. Journal of the Saudi Society of Agricultural Sciences, 15(2): 99-111.
Teshfam, M., Saeidi, J. and Zarei, A. (2010). Morphological and enzymological studies of the small intestine villi of rats receiving diets containing different levels of protein. Journal of Applied Animal Research, 37(2): 207-211.
Thavarajah, R., Mudimbaimannar, V.K., Elizabeth, J., Rao, U.K. and Ranganathan, K. (2012). Chemical and physical basics of routine formaldehyde fixation. Journal of Oral and Maxillofacial Pathology, 16(3): 400-405.
Tu, L., Yu, L. and Zhang, H. (2011). Morphology of rat testis preserved in three different fixatives. Journal of Huazhong University of Science and Technology [Medical Sciences], 31(2): 178-180.
Van Leeuwen, P., Mouwen, J.M.V.M., Van Der Klis, J.D. and Verstegen, M.W.A. (2004). Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance. British Poultry Science, 45(1): 41-48.
Williams, J.M., Duckworth, C.A., Vowell, K., Burkitt, M.D. and Pritchard, D.M. (2016). Intestinal preparation techniques for histological analysis in the mouse. Current Protocols in Mouse Biology, 6(2): 148-168.
Yegani, M. and Korver, D.R. (2008). Factors affecting intestinal health in poultry. Poultry Science, 87(10): 2052-2063.
