吸入类药物临床前研究中鼻组织脱钙技术优化：用于大鼠鼻黏膜组织病理学检查

Optimization of Nasal Tissue Decalcification Technique in Preclinical Studies of Inhaled Drugs: Histopathological Examination of Nasal Mucosa in Rats

关键词:  鼻  脱钙  骨组织  大鼠  组织病理学  吸入类药物

Key words:  nose  decalcification  bone tissue  rat  histopathological  inhaled drugs

摘要:

目的 研究不同脱钙液对大鼠鼻组织的脱钙作用及其病理切片染色效果。方法 选择10%乙二胺四乙酸（ethylene diamine tetraacetic acid，EDTA）、10%甲酸、5%硝酸脱钙液3种不同脱钙液，在室温静置及微波条件下，对大鼠鼻组织的脱钙时间和脱钙效果进行比较分析，综合评估骨组织经不同脱钙方法后制成病理切片质量。结果 常温条件下鼻组织经过EDTA脱钙液所需脱钙时间最长，微波条件下鼻组织经硝酸脱钙液所需脱钙时间最短，经EDTA脱钙液的鼻组织切片质量、HE染色、MASSON染色和免疫组化染色中质量最佳，硝酸脱钙液的鼻组织的切片质量最差，甲酸脱钙液的鼻组织HE染色效果较佳，MASSON和免疫组化染色质量略差。结论 EDTA脱钙液配合微波进行的鼻组织脱钙，脱钙效率明显提升，且切片和染色效果俱佳。

Abstract:

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.