Histopathologic Evaluation of GA Aerosol Effects

Morphological changes caused by GA aerosols were evaluated at T5 and PT7. Sample preparation and histopathology staining were performed as described previously [12 (link),13 (link)]. Hematoxylin and eosin (H&E) staining was conducted using a Leica Autostainer (Buffalo Grove, IL, USA); histopathologic changes were evaluated and scored by a board-certified pathologist. Digital images of the immunohistochemistry (IHC)-stained sections were obtained by scanning with the Aperio Scanscope System (Leica Biosystems, Vista, CA, USA). The percentages of cleaved caspase-3-positive apoptotic bodies, i.e., apoptotic index (AI), and periodic acid-Schiff (PAS)-stained goblet cells were calculated semi-automatically; the total numbers of nuclei were quantified automatically with the Nuclear Algorithm (Aperio Scanscope System), which counts the numbers of positive (brown) and negative (blue) nuclei in the section under examination, and the apoptotic bodies and PAS-stained goblet cells were counted manually. The percentage of Ki-67-positive nuclei, i.e., proliferative index (PI), was evaluated automatically with the Nuclear Algorithm.