Experimental and Toxicologic Pathology 66 (2014) 469–475 Contents lists available at ScienceDirect Experimental and Toxicologic Pathology j ourna l h omepage: www.elsevier.de/etp Efficiency of different decalcification protocols for nasal osseous structures in a rat experimental model of allergic rhinitis, and their effects on epithelial histology: An attempt at standardization George V. Guibas a,b,∗ , Sotiris Lakis c , Christoforos Gkimpas b , Marianthi Manda b , Dorothea Kapoukranidou d , Evangelia Spandou b a Allergy Department, 2nd Pediatric Clinic, University of Athens, Greece b Laboratory of Experimental Physiology, Aristotle University of Thessaloniki, Greece c Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki, Greece d Laboratory of Physiology, Aristotle University of Thessaloniki, Greece a r t i c l e i n f o Article history: Received 25 July 2014 Accepted 3 September 2014 Keywords: Decalcification Allergic rhinitis Experimental models Nasal structures Respiratory epithelium Histology a b s t r a c t Introduction: Decalcification of osseous specimens is required for histological analysis; this however may cause tissue damage. In rodent models of allergic rhinitis (AR), epithelial histologic assessment necessitates prior decalcification of the nasal osseous structures. However, respiratory epithelium is highly susceptible to damage, and rat nasal architecture is elaborate and its sectioning is challenging. Nevertheless, decalcification is not standardized in experimental AR. We therefore undertook this task, in order to reduce experimental bias. Methods: Six-to-eight week-old Wistar rats underwent an AR protocol. Subsequently, nasal structures were decalcified in the following mediums: (i) formic acid 10% for 5 and 20 days; (ii) formic acid 15% for 5 and 15 days; (iii) Morse Solution for 5 and 20 days and (iv) EDTA for 20 and 40 days. Decalcification efficiency/speed was evaluated via radiographic analysis. Furthermore, specimens were stained with hematoxylin and eosin and assessed for preservation of epithelial features. Results: Specimens were appropriately decalcified in 5 days in the formic acid-based mediums and in 20 days in EDTA with minimal epithelial damage. EDTA for 40 days had no unacceptable adverse effects; conversely, 15 and/or 20 days in acid-based agents provided no extra benefit for decalcification and were detrimental to the epithelium. Conclusions: EDTA treatment for 20 days is appropriate for decalcification of nasal structures in rat models of allergic rhinitis; further incubation preserves epithelial integrity but is not required. When urgency is a factor, formic-acid-based decalcification for 5 days yields acceptable results. © 2014 Elsevier GmbH. All rights reserved. 1. Introduction Bone-containing specimens require treatment with calcium- reacting agents, that is, acids which turn insoluble calcium salts into soluble, and/or chelators which take up calcium ions (Alers et al., 1999; Mattuella et al., 2007). Acids decalcify rapidly, but deteriora- tion of tissue staining can occur after long incubation (Alers et al., 1999; Begum et al., 2010). Conversely, chelators have little effect on ∗ Corresponding author at: Allergy Department and Allergy Research Center, 41 Fidippidou Street, Athens 115 27, Greece. Tel.: +30 6976552831; fax: +30 2107777693. E-mail address: george.guibas@gmail.com (G.V. Guibas). non-osseous tissues, but decalcification is slow (Yamamoto-Fukud et al., 2000). In experimental rodent protocols of allergic rhinitis (AR), decal- cification of the nasal bone is required. Nevertheless, decalcification techniques have not been standardized in this setting: as they are routine laboratory practices, they are not usually questioned in regard to modifications that could lead to higher tissue quality (Fernandes et al., 2007). However, tissue quality is especially impor- tant in models of AR, as the subtlety of pathologic alterations may cause their obscuring by decalcification-induced epithelial damage. Alongside these disease-specific issues, there are also anatomy- related ones: rat nose has numerous and elaborate turbinates lined by a plethora of epithelial types that occupy distinct areas (Harkema et al., 2006). For instance, olfactory epithelium covers approxi- mately 50% of the nasal cavity, whereas transitional epithelium is http://dx.doi.org/10.1016/j.etp.2014.09.001 0940-2993/© 2014 Elsevier GmbH. All rights reserved.