Fig. 1

HBOT synchronously increases ISCs proliferation. A Schematic workflow of HBOT treatment and experimental procedures on murine small intestine samples. B-E) Representative immunofluorescence images of ISC proliferation in intestinal crypts. B Proliferative cells were detected by BrdU labeling (Red). C ISCs labeled as Olfm4 + cells, a cytoplasmatic marker (Green). The inset shows DAPI nuclear staining (Blue). D Merged image. The yellow box indicates a crypt with proliferating ISCs among other cell populations. Bar = 15 µm. E Zoom of the yellow box from image D Red arrowheads indicate proliferating ISCs while white arrowheads indicate other proliferating cells within the intestinal crypt. Bar = 30 µm. F–G Representative immunohistochemistry images of intestinal crypts with proliferating BrdU + cells; experimental groups as indicated. Nuclei labeled by hematoxylin counterstaining. Bar = 20 µm. Yellow dotted lines outline the crypts while white arrowheads show BrdU + cells. J Quantification of BrdU + cells per crypt for each treatment. Asterisks indicate statistical significance. Permutation test. < 0.05. n = 5. K Stabilization coefficient indicating the variation level within each group. Only H20D shows significant differences, indicating synchronicity. Permutation test. < 0.05. n = 4. L, M Representative immunofluorescence images of control and HBOT intestinal crypts. The dashed line shows cellular limits. White arrowheads show cells positive for both BrdU and Olfm4 markers. Bar = 25 µm. N Quantification of BrdU + and Olfm4 + cells within the crypt between control and HBOT groups (for 20 days). n = 3. Asterisks indicate statistical significance. < 0.05