Hydrogen gas and preservation of intestinal stem cells in mesenteric ischemia and reperfusion

Figure 1 Intestinal mucosal injury with ischemia-reperfusion injury. A: Histological sections with H&E stain showed pyknosis in the epithelial layer in the ischemia group; B: Extensive pyknosis in the epithelial layer, denudation of the tip of the villi, and capillary congestion in the reperfusion group; C: Mild epithelial pyknosis and denudation of the villi in the hydrogen group. Scale bar: 50 μm.

Figure 2 Intestinal stem cell injury by ischemia-reperfusion injury. A: Immunofluorescence staining with caspase-3 (green) revealed mucosal injury at the epithelial layer closed to the tip of the villi in the ischemia group; B: Extensive apoptosis was identified at the crypt base and the whole epithelial layer in the reperfusion group; C: Apoptosis at the crypt base was limited in the hydrogen group. Scale bar: 100 μm.

Figure 3 Intestinal stem cell with double immunofluorescence staining. A: Double immunofluorescence staining with caspase-3 (red) and leucine-rich repeat-containing G-protein-coupled 5 (LGR5) (green) showed that LGR5-positive cells at the crypt base did not undergo apoptosis in the ischemia group (caspase-3 and LGR5 were separately stained); B: Multiple LGR5-positive cells underwent apoptosis after reperfusion (there were double-stained cells with caspase-3 and LGR5). Scale bar: 50 μm.

Figure 4 Quantitative analysis of RNA. A: Leucine-rich repeat-containing G-protein-coupled 5 expression was significantly lower in the reperfusion group than in the ischemia group, but it was significantly higher in the hydrogen group than in the reperfusion group. Olfactomedin 4 expression was also relatively higher in the hydrogen group than in the reperfusion group, although the differences were not significant; B: Hes1 expression was relatively high in the reperfusion group than in the ischemia and hydrogen groups, whereas Jag2 and Notch1 expressions were comparable between the ischemia, reperfusion, and hydrogen groups. ^aP < 0.05. LGR5: Leucine-rich repeat-containing G-protein-coupled 5; OLFM4: Olfactomedin 4; Hes1: Hairy and enhancer of split 1; Jag2: Jagged 2; Notch1: Neurogenic locus notch homolog protein 1.

Figure 5 Mucosal oxidative injury in the ischemia-reperfusion model. A: Immunostaining for 8-hydroxy-2'-deoxyguanosine showed limited oxidative stress at the mucosa in the ischemia group, although there was considerable pyknosis at the tip of the villi; B: Extensive oxidative injury throughout the epithelium, including the crypt base, in the reperfusion group; C: Mild-to-moderate oxidative injury at the mucosa of the crypt base, along with pyknosis with the denudation at the tip of the villi, in the hydrogen group. Scale bar: 50 μm.

Figure 6 Systematic oxidative injury in the ischemia-reperfusion model. Serum 8-hydroxy-2'-deoxyguanosine (8-OHdG) concentration immediately after each intervention (ischemia, ischemia-reperfusion, and ischemia-reperfusion under hydrogen gas inhalation) was measured. Although not significant, the 8-OHdG concentration was higher in the reperfusion and hydrogen groups than in the ischemia group. 8-OHdG: 8-hydroxy-2'-deoxyguanosine.