title: 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.
abstract: BACKGROUND
  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.
authors.affiliation: Allergy Department, 2nd Pediatric Clinic, University of Athens, Greece; Laboratory of Experimental Physiology, Aristotle University of Thessaloniki, Greece. Electronic address: george.guibas@gmail.com.
authors.forename: George V
authors.initials: GV
authors.lastname: Guibas
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki, Greece.
authors.forename: Sotiris
authors.initials: S
authors.lastname: Lakis
authors.affiliation: Laboratory of Experimental Physiology, Aristotle University of Thessaloniki, Greece.
authors.forename: Christoforos
authors.initials: C
authors.lastname: Gkimpas
authors.affiliation: Laboratory of Experimental Physiology, Aristotle University of Thessaloniki, Greece.
authors.forename: Marianthi
authors.initials: M
authors.lastname: Manda
authors.affiliation: Laboratory of Physiology, Aristotle University of Thessaloniki, Greece.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Kapoukranidou
authors.affiliation: Laboratory of Experimental Physiology, Aristotle University of Thessaloniki, Greece.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Spandou
mesh_terms: D000818:Animals; D001842:Bone and Bones; D003648:Decalcification Technique / Q000592:standards*; D004195:Disease Models, Animal; D008297:Male; D009666:Nose; D051381:Rats; D017208:Rats, Wistar; D065631:Rhinitis, Allergic / Q000473:pathology*
keywords: Allergic rhinitis; Decalcification; Experimental models; Histology; Nasal structures; Respiratory epithelium