The effects of hydrogen on ventilator-induced lung injury (VILI)-associated histopathological changes by mechanical ventilation with low tidal volume (10 mL/kg). (a) Representative images of lungs stained with F4/80 to visualize macrophages. Left panel: There were few F4/80-positive cells in the lungs of sham control mice. Middle panel: A marked increase in F4/80-postive cells was observed in the lung with low-tidal volume ventilation. Right panel: Ventilation with 2% hydrogen significantly reduced the number of macrophages. Magnification = 600 ×. Representative images from four animals for each experimental group are shown. White arrowheads indicate F4/80-positive cells. (b) Bronchial cell apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assay. Left panel: Few apoptotic cells were seen in the bronchial epithelium of sham control mice. Middle panel: There was an increase in the number of TUNEL-positive cells in the bronchial epithelium in the lung with VILI in 2% nitrogen. Right panel: Hydrogen therapy decreased the number of TUNEL-positive cells. Magnification = 800 ×. Representative images from four animals for each experimental group are shown. Black arrowheads indicate TUNEL-positive cells. (c) The positive cells were counted in a blinded manner and expressed as the number per high-power field (HPF). Histograms indicate the number of F4/80-positive cells/HPF and TUNEL-positive epithelial cells/HPF. N = 4. *P < 0.05 versus sham/N2 and sham/H2; #P < 0.05 versus VILI/N2.