Optimization of Nasal Tissue Decalcification Technique in Preclinical Studies of Inhaled Drugs: Histopathological Examination of Nasal Mucosa in Rats
WANG Yu
;
LAN Xiuhua
;
SHEN Bin
;
GAO Dan
;
FENG Zhen
Chinese Journal of Modern Applied Pharmacy.2023;40(20):2846-2850. doi: 10.13748/j.cnki.issn1007-7693.20231880
OBJECTIVE New inhaled formulations that act on the nose, mouth, respiratory tract, and whole body have received increasing attention. Meanwhile, the research and declaration of inhaled drugs have become hot spots amid infectious respiratory pandemic diseases worldwide. Due to the special anatomic structure of the nose, folds, grooves, and special structures may cause the specific uptake and deposition of inhaled substances. There are various epithelial tissues, glands, muscles, and cartilages in the vestibule, respiratory, and olfactory parts of the nose. Inhaled substances can generate irritating and toxic effects on various parts. The pathological diagnosis results from the preclinical safety evaluation of inhaled drugs are considered the gold standard for judging drug toxicology. The nose is composed of many bone components, and decalcification is required for the sectioning of hard bone tissues. Therefore, an efficient and high-quality decalcification method is the crucial pathological technique for evaluating inhaled drugs. METHODS In this study, 10% ethylenediamine tetraacetic acid(EDTA), 10% formic acid, and 5% nitric acid decalcification solutions were selected. Besides, the decalcification time and effect of these decalcification solutions for rat nasal tissues were compared and analyzed under static room temperature and microwave conditions. Moreover, the quality of pathological bone tissue sections prepared through different decalcification methods was comprehensively evaluated. RESULTS Compared with the decalcification method under normal temperature, the decalcification time under the treatment of KOS decreased significantly. The treatment with the EDTA decalcification solution had the longest decalcification time under normal temperature, while the treatment with the nitric acid decalcification solution had the shortest decalcification time under microwaves. During section evaluation, the EDTA decalcification solution had a higher quality score under normal temperature and microwaves, which indicated that the section quality was favorable. The nitric acid decalcification solution had a lower section quality score under microwaves, which indicated that the section quality was unfavorable. There was medium section quality for the formic acid decalcification solution under microwaves and normal temperature and for the nitric acid decalcification solution under normal temperature. The HE staining results suggested that there were incomplete nasal mucosa epithelia, fragmentation, and pink nasal bone tissues in the tissue sections treated by the nitric acid decalcification solution, presenting a peracid state. In the tissue sections treated by the formic acid decalcification solution and the EDTA decalcification solution, the nucleus of epithelial cells was blue-purple, the cytoplasm and interstitial components were pink, and the epithelial tissue structure of nasal mucosa was intact. The MASSON staining results suggested that in the tissue sections treated by the nitric acid decalcification solution, the whole section staining was red, the positive area was not obvious, and the epithelial cell differentiation was not prominent, with a fuzzy structure. In the tissue sections treated by the formic acid decalcification solution, the sections were slightly detached during staining, and slight cracks were observed in submucosa tissues. In the tissue sections treated by the EDTA decalcification solution, the structure of positive regions and epithelial mucosa regions was clear, and the nuclear and interstitial components were clearly distinguished. The immunohistochemical staining (Ki67) results suggested that in the tissue sections treated by the nitric acid decalcification solution, the staining of positive regions was uneven, and there were nonspecific negative reactions in some regions. In addition, local epithelial cells were unstained. In the tissue sections treated by the formic acid decalcification solution, the local regions were not clearly stained, and nonspecific negative and positive reactions appeared in some local regions. In the tissue sections treated by the EDTA decalcification solution, the positive regions were prominent, the boundaries between negative regions and positive ones were clear, and each region of the sections was stained evenly. CONCLUSION Among the three decalcification solutions in this study, the nitric acid decalcification solution had the shortest decalcification time while the poor section and staining quality. The decalcification time of nasal tissues through the EDTA decalcification solution combined with microwaves was significantly shorter than that through the EDTA decalcification solution at normal temperature. Furthermore, this decalcification method achieved favorable section and staining quality.
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