Fig. 1Schematic drawing of terminal deoxynucleotidyl transferase dUTP nick end labeling staining on the light microscopy (LM) and electron microscopic (EM). DAB, diaminobenzidine.
Fig. 2Gross (A-C) and microscopic (D-I) features of lungs exposed to sidestream smoke. (A) Group 1 (1-mo exposure to three cigarettes a day). (B) Group 3 (1-mo exposure to seven cigarettes a day). (C) Group 4 (3-mo exposure to five cigarettes a day). (D-F) Groups 2 (E) and 3 (F) show more prominent alveolar collapse and focal emphysematous change than the group 1 (D). (G-I) Group 2 (G) shows mild bronchiolar epithelial proliferation. Group 4 (H) shows bronchiolar epithelial cell proliferation, peribronchiolar inflammatory cell infiltration, and focal emphysematous change. Group 5 (I) shows peribronchiolar inflammatory thickening, and adjacent alveolar patchy emphysematous change.
Fig. 3Ultrastructural changes in lungs exposed to sidestream smoke. (A-C) Scanning electron microscopy (SEM) of the bronchiolar mucosa shows a marked destruction and a loss of the cilia of the epithelial cells, and an irregular distribution of the Clara cells in groups 4 (B) and 5 (C), compared to group 1 (A). (D-F) Transmission electron microscopy of the alveolar regions shows more prominent alveolar wall destruction with epithelial cell injury, cytoplasmic bleb formation (black arrow), and free-floating cellular debris (white arrows) in groups 2 (E) and 3 (F), compared to group 1 (D). (G-I) SEM of the alveolar walls shows more patchy collapse, irregular dilatation and wall thickening in groups 2 (H) and 3 (I) than group 1 (G) (A-C, ×25,000; D, ×3,000; E, F, ×8,000; G-I, ×25,000).
Fig. 4Immunohistochemical expression of tumor necrosis factor α (TNF-α), tumor growth factor β1 (TGF-β1), cytokeratin 14 (CK14), and Ki-67 in rats which are exposed to sidestream smoke. TNF-α is not expressed in lungs of group 1 (A). But there is a multiple patchy expression of TNF-α in group 4 (B) and a diffuse expression along the alveolar epithelium in group 5 (C). TGF-β1 is expressed in alveolar macrophages but not in alveolar cells in group 1 (D). In group 2, there is a multi-focal expression of TGF-β1 in the alveolar endothelial and epithelial cells (E). In group 4, there is a diffuse expression of TGF-β1 along the alveolar endothelial and epithelial cells (F). CK14 staining is diffuse in the bronchiolar epithelial and basal cells of groups 1 (G) and 4 (H). Ki-67 is focally expressed in the alveolar epithelial cells in group 4 (I).
Fig. 5Light microscopy (LM) and electron microscopic (EM) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptotic cells. (A-C) LM TUNEL stains show a few scattered apoptotic nuclei in group 1 (A), more scattered apoptotic nuclei in group 3 (B), and frequent apoptotic alveolar macrophages (arrows) in group 4 (C). (D-F) EM TUNEL stains show moderately electron dense apoptotic nuclei in the alveolar epithelial and endothelial cells. Apoptotic nuclei are seen more frequently in groups 3 (E) and 4 (F) than group 2 (D). (D, ×12,000; E, ×18,000; F, ×11,000)
Fig. 6Western blot analysis of proform-poly (ADP-ribose) polymerase (PARP). Expression of proform-PARP is correlated with the volume of exposure to sidestream smoke in groups 1, 2, and 3. The degree of the expression of proform-PARP is also decreased following a long-term exposure to sidestream smoke in groups 4 and 5 as compared with the control group. PARP, proform-PARP; lane 1 and 5, control group; lane 2, group 1; lane 3, group 2; lane 4, group 3; lane 6, group 4; lane 7, group 5.
Table 1Primary antibodies used for immunohistochemical staining
Table 2Ultrastructural changes in bronchiolar and alveolar cells depending on the volume of smoke exposure
Table 3Ultrastructural changes in bronchiolar and alveolar cells depending on the duration of smoke exposure
Table 4Scoring of immunohistochemical expression of alveolar and bronchiolar cells