The remaining 6 mice in each group were used for pathological examination. The lungs were fixed by a 10% neutral phosphate-buffered formalin solution. After separation of the lobes, 2-mm-thick blocks were taken for paraffin embedding. Embedded blocks were sectioned at a thickness of 3 μm and then stained with hematoxylin and eosin (H & E) to evaluate the degree of infiltration of eosinophils and lymphocytes in the airway from proximal to distal. The sections were stained with periodic acid-Schiff (PAS) to evaluate the degree of proliferation of goblet cells in the bronchial epithelium. A pathological analysis of inflammatory cells and epithelial cells in the airway was performed using a Nikon ECLIPSE light microscope (Nikon Co., Tokyo, Japan). The degree of infiltration of eosinophils and lymphocytes in the airway or proliferation of goblet cells in the bronchial epithelium was graded in blinded fashion as: 0, not present; 1, slight; 2, mild; 3, moderate; 4, moderate to marked; 5, marked. ‘Slight’ was defined as less than 20% of the airway with eosinophilic inflammatory reaction or with goblet cells stained with PAS; ‘mild’ as 21 - 40%; ‘moderate’ as 41 - 60%; ‘moderate to marked’ as 61 - 80%; and marked as more than 80% of the airway
[22 (link), 23 (link)].
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