Analysis of Uterine Morphology in Ovariectomized Rats Treated With Alendronate and Hop Extract Using Open-Source Software

Edi Rođak; Nada Oršolić; Robert Grgac; Jasmina Rajc; Marina Bakula; Nikola Bijelić

doi:10.5566/ias.2791

Authors

Edi Rođak Department for Histology and Embryology, Faculty of Medicine Osijek, University of Osijek, J. Huttlera 4, 31000 Osijek, Croatia https://orcid.org/0000-0001-7814-2031
Nada Oršolić Department of Animal Physiology, Faculty of Science Zagreb, University of Zagreb, Rooseveltov trg 6, Zagreb, Croatia https://orcid.org/0000-0001-5102-3606
Robert Grgac Institute of Entomology, Biology Centre, Czech Academy of Sciences, Branišovská 1160/31, 37005 České Budějovice, Czechia Faculty of Science, University of South Bohemia, Branišovská 1645/31a, 37005 České Budějovice, Czechia https://orcid.org/0000-0001-9589-1472
Jasmina Rajc Department of Pathology and Forensic Medicine, University Hospital Centre Osijek, J. Huttlera 4, 31000 Osijek, Croatia Department of Pathology and Forensic Medicine, Faculty of Medicine Osijek, University of Osijek, J. Huttlera 4, 31000 Osijek, Croatia https://orcid.org/0000-0003-4007-8390
Marina Bakula Department of Pathology and Forensic Medicine, University Hospital Centre Osijek, J. Huttlera 4, 31000 Osijek, Croatia
Nikola Bijelić Department for Histology and Embryology, Faculty of Medicine Osijek, University of Osijek, J. Huttlera 4, 31000 Osijek, Croatia https://orcid.org/0000-0003-4136-820X

DOI:

https://doi.org/10.5566/ias.2791

Keywords:

alendronate, hop extract, image analysis, open-source, osteoporosis, uterus

Abstract

Free and open-source software for image analysis and morphological measurements in scientific research is rising in popularity and capabilities as new methods, plugins and macros are being actively developed. A semi-automated method for measuring rat uterus morphology using free and open-source software (Gimp and FIJI) is demonstrated in this paper. Research was performed on ovariectomized rats as a model of osteoporosis (with sham-operated control group). The animals were treated with alendronate, hop extract or the combination of the two. Whole histological slides were photographed and images were manually pre-processed in Gimp. Color masks from Gimp were loaded in FIJI and polar transformation and measurements were made using a custom macro. This analysis was supplemented by manual assessment of Ki67 proliferation marker expression by a pathologist. Our results suggest that monotherapy or combination therapy with alendronate and hop extract does not cause proliferation of the endometrium in ovariectomized rats and would be safe for use in osteoporosis treatment in this regard. The semi-automated method used in this research is more precise and unbiased than older manual methods. Furthermore, it can be easily adapted for analysis of whole-slide images of almost any round or oval organ.

References

Aeffner F, Zarella MD, Buchbinder N, Bui MM, Goodman MR, Hartman DJ, Lujan GM, Molani MA, Parwani A V., Lillard K, Turner OC, Vemuri VNP, Yuil-Valdes AG, Bowman D (2019). Introduction to Digital Image Analysis in Whole-slide Imaging: A White Paper from the Digital Pathology Association. J Pathol Inform 10:9. https://doi.org/10.4103/jpi.jpi_82_18

An K-C (2016). Selective Estrogen Receptor Modulators. Asian Spine J 10:787–91. https://doi.org/10.4184/asj.2016.10.4.787

Bijelić N, Belovari T, Stolnik D, Lovrić I, Baus Lončar M (2017). Histomorphometric Parameters of the Growth Plate and Trabecular Bone in Wild-Type and Trefoil Factor Family 3 (Tff3)-Deficient Mice Analyzed by Free and Open-Source Image Processing Software. Microsc Microanal 23:818–25. https://doi.org/10.1017/S1431927617000630

Bijelić N, Kozina N, Rođak E, Bazina I, Šešelja K, Baus Lončar M, Belovari T, Jukić I, Drenjančević I (2022). Structural characteristics of femoral arteries in wild-type and TFF3 knock-out mice on standard and high-salt diet. In 4th Croatian Microscopy Congress with international participation (eds. J. Macan, G. Kovačević), pp. 34–5. Poreč, Hrvatska. Retrieved from https://www.bib.irb.hr/1196468

Boyaci C, Sun W, Robertson S, Acs B, Hartman J (2021). Independent Clinical Validation of the Automated Ki67 Scoring Guideline from the International Ki67 in Breast Cancer Working Group. Biomolecules 11:1612. https://doi.org/10.3390/biom11111612

Burel JM, Besson S, Blackburn C, Carroll M, Ferguson RK, Flynn H, Gillen K, Leigh R, Li S, Lindner D, Linkert M, Moore WJ, Ramalingam B, Rozbicki E, Tarkowska A, Walczysko P, Allan C, Moore J, Swedlow JR (2015). Publishing and sharing multi-dimensional image data with OMERO. Mamm Genome 26:441–7. https://doi.org/10.1007/S00335-015-9587-6/FIGURES/1

Burton JL, Wells M (2002). The effect of phytoestrogens on the female genital tract. J Clin Pathol 55:401–7. https://doi.org/10.1136/jcp.55.6.401

Chen M-N, Lin C-C, Liu C-F (2015). Efficacy of phytoestrogens for menopausal symptoms: a meta-analysis and systematic review. Climacteric 18:260–9. https://doi.org/10.3109/13697137.2014.966241

Christoffel J, Rimoldi G, Wuttke W (2006). Effects of 8-prenylnaringenin on the hypothalamo-pituitary-uterine axis in rats after 3-month treatment. J Endocrinol 188:397–405. https://doi.org/10.1677/joe.1.06384

Dobbs MB, Buckwalter J, Saltzman C (1999). Osteoporosis: the increasing role of the orthopaedist. Iowa Orthop J 19:43–52.

Drake MT, Clarke BL, Khosla S (2008). Bisphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc 83:1032–45. https://doi.org/10.4065/83.9.1032

Ensrud KE, Crandall CJ (2017). Osteoporosis. Ann Intern Med 167:ITC17–ITC32. https://doi.org/10.7326/AITC201708010

Feng M, Deng Y, Yang L, Jing Q, Zhang Z, Xu L, Wei X, Zhou Y, Wu D, Xiang F, Wang Y, Bao J, Bu H (2020). Automated quantitative analysis of Ki-67 staining and HE images recognition and registration based on whole tissue sections in breast carcinoma. Diagn Pathol 15:65. https://doi.org/10.1186/s13000-020-00957-5

Fu S, Zeng G, Zong S, Zhang Z, Zou B, Fang Y, Lu L, Xiao D (2014). Systematic review and meta-analysis of the bone protective effect of phytoestrogens on osteoporosis in ovariectomized rats. Nutr Res 34:467–77. https://doi.org/10.1016/j.nutres.2014.05.003

Guo J, Nikolic D, Chadwick LR, Pauli GF, van Breemen RB (2006). Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.). Drug Metab Dispos 34:1152–9. https://doi.org/10.1124/dmd.105.008250

Hammer DAT, Ryan PD, Hammer Ø, Harper DAT (2001). Past: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontol Electron 4:178. Retrieved from http://palaeo-electronica.orghttp//palaeo-electronica.org/2001_1/past/issue1_01.htm.

Hümpel M, Isaksson P, Schaefer O, Kaufmann U, Ciana P, Maggi A, Schleuning W-D (2005). Tissue specificity of 8-prenylnaringenin: protection from ovariectomy induced bone loss with minimal trophic effects on the uterus. J Steroid Biochem Mol Biol 97:299–305. https://doi.org/10.1016/j.jsbmb.2005.05.009

Ji M-X, Yu Q (2015). Primary osteoporosis in postmenopausal women. Chronic Dis Transl Med 1:9–13. https://doi.org/10.1016/j.cdtm.2015.02.006

Kassab A (2019). Effect of Fosamax on the duodenal mucosa in adult male albino rats and the possible protection by nigella sativa oil: a histological and immunohistochemical study. Egypt J Histol 42:0-0. https://doi.org/10.21608/ejh.2019.10902.1105

Keiler AM, Dörfelt P, Chatterjee N, Helle J, Bader MI, Vollmer G, Kretzschmar G, Kuhlee F, Thieme D, Zierau O (2015). Assessment of the effects of naringenin-type flavanones in uterus and vagina. J Steroid Biochem Mol Biol 145:49–57. https://doi.org/10.1016/j.jsbmb.2014.10.001

Kinra P, Malik A (2020). Ki 67: Are we counting it right? Indian J Pathol Microbiol 63:98–9. https://doi.org/10.4103/IJPM.IJPM_770_19

Kitson S, Sivalingam VN, Bolton J, McVey R, Nickkho-Amiry M, Powell ME, Leary A, Nijman HW, Nout RA, Bosse T, Renehan AG, Kitchener HC, Edmondson RJ, Crosbie EJ (2017). Ki-67 in endometrial cancer: scoring optimization and prognostic relevance for window studies. Mod Pathol 30:459–68. https://doi.org/10.1038/modpathol.2016.203

Kozina N, Mihaljević Z, Lončar MB, Mihalj M, Mišir M, Radmilović MD, Justić H, Gajović S, Šešelja K, Bazina I, Horvatić A, Matić A, Bijelić N, Rođak E, Drenjančević I, Jukić I (2019). Impact of High Salt Diet on Cerebral Vascular Function and Stroke in Tff3−/−/C57BL/6N Knockout and WT (C57BL/6N) Control Mice. Int J Mol Sci 2019, Vol 20, Page 5188 20:5188. https://doi.org/10.3390/IJMS20205188

Luo D, Kang L, Ma Y, Chen H, Kuang H, Huang Q, He M, Peng W (2014). Effects and mechanisms of 8-prenylnaringenin on osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7. Food Sci Nutr 2:341–50. https://doi.org/10.1002/fsn3.109

Marusak RA, Radi ZA, Obert L (2007). Expression of Ki-67 in the uterus during various stages of the estrous cycle in rats. Exp Toxicol Pathol 59:151–5. https://doi.org/10.1016/j.etp.2007.06.004

Ming L-G, Lv X, Ma X-N, Ge B-F, Zhen P, Song P, Zhou J, Ma H-P, Xian CJ, Chen K-M (2013). The prenyl group contributes to activities of phytoestrogen 8-prenynaringenin in enhancing bone formation and inhibiting bone resorption in vitro. Endocrinology 154:1202–14. https://doi.org/10.1210/en.2012-2086

National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. (2011). Guide for the Care and Use of Laboratory Animals. National Academies Press, Washington, D.C. https://doi.org/10.17226/12910

Newcomb PA, Passarelli MN, Phipps AI, Anderson GL, Wactawski-Wende J, Ho GYF, O’Sullivan MJ, Chlebowski RT (2015). Oral bisphosphonate use and risk of postmenopausal endometrial cancer. J Clin Oncol 33:1186–90. https://doi.org/10.1200/JCO.2014.58.6842

Pohjanvirta R, Nasri A (2022). The Potent Phytoestrogen 8-Prenylnaringenin: A Friend or a Foe? Int J Mol Sci 23:3168. https://doi.org/10.3390/ijms23063168

Possemiers S, Bolca S, Grootaert C, Heyerick A, Decroos K, Dhooge W, De Keukeleire D, Rabot S, Verstraete W, Van de Wiele T (2006). The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine. J Nutr 136:1862–7. https://doi.org/10.1093/jn/136.7.1862

Quarmby VE, Korach KS (1984). The influence of 17 beta-estradiol on patterns of cell division in the uterus. Endocrinology 114:694–702. https://doi.org/10.1210/endo-114-3-694

Rimoldi G, Christoffel J, Wuttke W (2006). Morphologic changes induced by oral long-term treatment with 8-prenylnaringenin in the uterus, vagina, and mammary gland of castrated rats. Menopause 13:669–77. https://doi.org/10.1097/01.gme.0000196596.90076.d0

Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:1–26. https://doi.org/10.1186/S12859-017-1934-Z/FIGURES/7

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–82. https://doi.org/10.1038/nmeth.2019

Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–5.

Sedgewick J (2017). Acquisition and Post-Processing of Immunohistochemical Images. In Methods in molecular biology (Clifton, N.J.), pp. 75–106. Methods Mol Biol. https://doi.org/10.1007/978-1-4939-6759-9_4

Sehmisch S, Hammer F, Christoffel J, Seidlova-Wuttke D, Tezval M, Wuttke W, Stuermer KM, Stuermer EK (2008). Comparison of the phytohormones genistein, resveratrol and 8-prenylnaringenin as agents for preventing osteoporosis. Planta Med 74:794–801. https://doi.org/10.1055/s-2008-1074550

Słupski W, Jawień P, Nowak B (2021). Botanicals in Postmenopausal Osteoporosis. Nutrients 13. https://doi.org/10.3390/nu13051609

Solomon RW (2009). Free and Open Source Software for the Manipulation of Digital Images. Am J Roentgenol 192:W330–4. https://doi.org/10.2214/AJR.08.2190

Soydan SS, Araz K, Senel F V., Yurtcu E, Helvacioglu F, Dagdeviren A, Tekindal MA, Sahin F (2015). Effects of alendronate and pamidronate on apoptosis and cell proliferation in cultured primary human gingival fibroblasts. Hum Exp Toxicol 34:1073–82. https://doi.org/10.1177/0960327115569808

Štulíková K, Karabín M, Nešpor J, Dostálek P (2018). Therapeutic Perspectives of 8-Prenylnaringenin, a Potent Phytoestrogen from Hops. Molecules 23. https://doi.org/10.3390/molecules23030660

Sung C-M, Kim RJ, Hah Y-S, Gwark J-Y, Park H Bin (2020). In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon. BMC Musculoskelet Disord 21:19. https://doi.org/10.1186/s12891-019-3014-1

Tella SH, Gallagher JC (2014). Prevention and treatment of postmenopausal osteoporosis. J Steroid Biochem Mol Biol 142:155–70. https://doi.org/10.1016/j.jsbmb.2013.09.008

Vesterinen T, Säilä J, Blom S, Pennanen M, Leijon H, Arola J (2022). Automated assessment of Ki-67 proliferation index in neuroendocrine tumors by deep learning. APMIS 130:11–20. https://doi.org/10.1111/apm.13190