Fig. 1
Persistent high concentration of H2 significantly accelerated cutaneous wound healing, blood perfusion and vessel formation. A Left: connection between each module in the H2 chamber system. Middle: Model of the full-thickness cutaneous wound in mouse backs; inside circle = original wound, outside circle = sampling boundary. Right: Scheme of a wound section after injury. B Timeline of animal experiments and daily H2 treatment (or under other conditions). Mice underwent surgery for 30 min and then immediately put in H2 chamber for 1 h. Thereafter, they were treated with H2 (or other conditions) every 24 h for 1 h until sacrifice. C Capture of wound area every two days after modeling in different persistent conditions (n = 5 for each group). D Comparison of wound closure percentage after modeling (persistent daily treatments). E H&E staining and Masson staining showing the tissue remodeling 11 days post-wounding (persistent daily treatments). F Treatment was applied for the first 3 days only, and the images of wound areas were captured every 2 days after modeling (days 0–9). G Comparison of wound closure percentage after modeling among the five groups (treatment for the first 3 days). H Blood flow perfusion 0–11 days post-wounding in the 66% H2, 5% H2, and Control groups. I Quantification of blood perfusion, SO2, tHb, and HHb in wound area among the three groups. J, K Representative immunofluorescence images and quantification for CD31+ (green) tube formation in dermal wounds of at day 11 post-wounding. L, M Panoramic scanning and the quantification of wound edge tube formation at the leading edge (L, 0–1 mm from wound site), mid-end (M, 1–2 mm from wound site) and distal (D, 2–3 mm from wound site) areas of the wound at day 3 post-wounding. Yellow dotted line indicates the boundary between the epithelium and dermis. N, O Representative microscopic images and the quantification of in vitro blood vessel formation of Human Umbilical Vein Endothelial Cells (HUVEC) at 12 h after different treatments. Red hatched line outlines the newly formed tubes. P GSEA Top-5 tube formation-related GO-bp enrichment plots showed the H2 accelerated tube formation (2 days earlier than the controls). Data in D, G, and I were analyzed by two-way ANOVA test, and data in K, M, and O were processed unpaired t test. All of the data are plotted as Mean ± SEM. *P value < 0.05; **P value < 0.01; ***P value < 0.001; no stars for P value > 0.05; *in D indicates a significant difference between the 66% H2 and control group, the 5% H2 and control group, separately; * in G indicates a significant difference between the 66% H2 and control group. Scale bar = 100 μm. Black dotted line in E indicates the boundary between the epithelium and dermis. Arrow in A (right) indicates the epithelial tongue; black arrowheads in E indicate blood capillaries in the dermal layer of the wound. b, basal layer; d, dermis; he, hypertrophic epidermal wound edge; hf, hair follicles; ife, interfollicular epithelium; s, scab; sm, smooth muscle; wb, wound bed