Fig. 5. Effect of H-MW-HA on chemotherapy-induced oral mucositis (CIOM) ulcer size percentage in 5-FU-treated mice.

Animals were sacrificed at different time points, and their tongues were collected for analysis. The harvested tongues were stained with 1% toluidine blue (TB) in 10% acetic acid. After imaging the tongues, the ulcer size and the total epithelialized upper surface area of the tongue were measured for all experimental groups (A–D). A Representative photographs showing the tongues of mice after staining with 1% toluidine blue (TB) in 10% acetic acid. The blue areas indicate CIOM ulcers at different surfaces of the tongue (white arrows). B Histological sections of the tongues stained with H&E in each experimental group. The black arrows indicate areas of erosion. C Representative photograph of a TB-stained tongue surface, highlighting the ulcerated epithelium (blue) on the upper surface of the tongue. For quantification, the ulcer size percentage of chemotherapy-induced oral mucositis (CIOM) relative to the total epithelialized surface of the tongue was measured using Fiji (ImageJ) software. The ulcer size percentage was calculated as the TB-positive surface area of the tongue (excluding excision trauma) divided by the total epithelialized surface area of the tongue. The numbered areas indicate the selected measured surface areas. D Mean percentage of oral mucositis (OM) ulcer size relative to the total epithelialized surface of the tongue at days 14, 16, and 19. E Mean percentage of OM ulcer size relative to the total epithelialized upper surface of the tongue on day 14. F Total mean percentage of OM ulcer size relative to the total epithelialized surface of the tongue between days 14 and 19. Data from days 14, 16, and 19 presented as the mean ± SEM. **p < 0.005 (n = 12, 12, 15, and 16 for groups A, B, C, and D, respectively).