Table 1.
Bacterial strains used to construct whole genomic DNA probes that were then used for testing hamster buccal epithelial cell samples.
Figure 1.
Real-time NO release profiles of the HPMC/GSNO formulations.
(A) Kinetic curves of NO release from the 0.5 mM and 2.0 mM HPMC/GSNO formulations, measured by chemiluminescence. (B) Integrated NO signals extracted from the curves of Fig. 1A, which indicate the total NO released from the formulations over the same time-scale. The straight lines denote linear regressions of the experimental data.
Figure 2.
Macroscopic and microscopic aspects of healthy hamster cheek pouches (a and b) or cheek pouches from animals subjected to 5-fluorouracil (5-FU)-induced oral mucositis and that received topical applications of the vehicle HPMC (c and d) or GSNO (0.5 mM) (e and f) observed on day 14.
Oral mucositis was induced by i.p. dministration of 5-fluorouracil followed by mechanical trauma of the cheek pouch. Animals received topical applications of S-nitrosoglutathione (0.5 mM HPMC/GSNO) or vehicle only (HPMC) at 1 h prior to 5-FU and every 12 h thereafter for 14 days. Each cheek pouch was everted and photographed, and samples were removed and processed for hematoxylin and eosin staining (100× magnification). Details: in Figure 2c, macroscopic edema (arrowhead) and vasodilation (dotted line) are shown; in Figure 2d, microscopic edema (larger circled) and severe vasodilation (smaller circled) are shown. Arrow = ulcerations.
Table 2.
Macroscopic and microscopic analysis of cheek pouch tissue of hamsters subjected to 5-FU-induced oral mucositis and treated with topical applications of S-nitrosoglutathione (0.5 mM), observed on days 10 and 14.
Figure 3.
TNF-α and IL-1β levels (pg/ml) in the cheek pouches of hamsters subjected to 5-FU-induced oral mucositis on day 14.
Oral mucositis was induced in hamsters by intraperitoneal (i.p.) injection of 5-FU followed by mechanical trauma (MT) of the cheek pouch. Animals received topical applications of a gel containing S-nitrosoglutathione (0.1, 0.5 and 2.0 mM HPMC/GSNO) 30 min prior to 5-FU and twice daily thereafter for 10 days or 14 days. Control groups comprised normal animals (N), animals subjected to mechanical trauma (MT) only and animals subjected to 5-FU-induced oral mucositis that received local application of saline (saline) or vehicle (HPMC). Bars denote the mean ± standard error of TNF-α and IL-1β levels in six animals per group. *denotes values significantly different (P<0.05) from the Healthy group; +denotes values significantly different (P<0.05) from the Saline group; #denotes values significantly different (P<0.05) from the HPMC group. Data were analyzed using the Kruskal Wallis and Mann Whitney tests.
Figure 4.
Representative examples of iNOS (1st row), IL-1β (2nd row), TNF-α (3rd row) and TGF-β RII (4th row) immunostaining on day 14 in tissues from cheek pouches of hamsters subjected to 5-FU-induced oral mucositis.
Staining was performed using cheek pouches from healthy animals (b, f, j, n) and animals subjected to 5-FU-induced mucositis that received topical applications of S-nitrosoglutathione (GSNO; 0.5 mM; d, h, l, p) or saline (c, g, k, o). Negative controls were samples of cheek pouches where the primary antibody was replaced with PBS-BSA (5%); no immunostaining was detected (a, e, i, m). Magnification, ×1000.
Figure 5.
Quantification of TNF-α- (A), IL-1β- (B), iNOS- (C) and TGF-β RII - (D) positive cells in cheek pouch tissues of hamsters subjected to 5-FU-induced oral mucositis, on day 14.
Oral mucositis was induced in hamsters by intraperitoneal (i.p.) injection of 5-FU followed by mechanical trauma (MT) of the cheek pouch. Animals received topical applications of a gel containing 0.5 mM S-nitrosoglutathione (GSNO) 30 min prior to 5-FU and twice daily thereafter for 10 days or 14 days. Control groups comprised healthy animals (H) and animals subjected to 5-FU-induced oral mucositis that received local applications of saline (Saline). Cells positive for staining were counted (10 fields per slide, 400×) for statistical comparisons. Bars denote the means ± standard errors of positive cells from four slides per group (4 animals per group). *denotes significant differences (P<0.05) compared with the Healthy group; +denotes a significant difference (P<0.05) compared with the Saline group; #denotes a significant difference (P<0.05) compared with the HPMC group. Data were analyzed using the Kruskal Wallis and Mann Whitney tests.
Figure 6.
Cell death and proliferation in the cheek pouches of hamsters subjected to 5-FU-induced oral mucositis, on day 14.
Oral mucositis was induced in hamsters by intraperitoneal (i.p.) injection of 5-FU followed by mechanical trauma (MT) of the cheek pouch. Animals received topical applications of a gel containing 0.5 mM S-nitrosoglutathione (GSNO) 30 min prior to 5-FU and twice daily thereafter for 10 days or 14 days. Control groups comprised healthy animals (H) and animals subjected to 5-FU-induced oral mucositis that received local applications of saline (saline). The TUNEL- and Ki67-positive cells were counted (10 fields per slide, 400×) for statistical comparisons. Bars denote the means ± standard errors of stained cells from at four slides per group (4 animals per group). *denotes a significant difference (P<0.05) compared with the Healthy group; **denotes a significant difference (P<0.05) compared with the MT group, +denotes a significant difference (P<0.05) compared with the Saline group; #denotes a significant difference (P<0.05) compared with the HPMC group. Data were analyzed using the Kruskal Wallis and Mann Whitney tests.
Figure 7.
Oral bacterial species evaluated in mucositis lesions of hamsters.
Only species showing significant differences between groups are displayed. Left panel: mean bacterial counts using log 10; Right panel: frequency of detection, %. * denotes p<0.05 and † p≤0.001 between groups using the Kruskal Wallis test. The number of animals in each group was at least five.
Figure 8.
Primary cultures of oral fibroblasts from newborn rats incubated for 90 min in medium containing GSNO (a–d) or GSNO-free (e–h).
The cells were immunostained using antibodies against smooth muscle anti-alpha-actin (c, d, g and h) or Ki67 (b, d, f and h), and nuclei were labeled with DAPI (a, d, e and h). Treatment of cells with 0.5 mM HPMC/GSNO for 90 min increased the expression of Ki67 (f and h) compared with untreated cells (b and d). Merged images are shown on the right (d and h). Scale bar = 10 µm.
Figure 9.
Quantification of immunohistochemical staining of oral myofibroblasts using an anti-Ki67 antibody.
A robust increase in Ki67-positive cells was observed following treatment with S-nitrosoglutathione (GSNO; 0.5 mM) for 90 and 180 min. Values denote the means ± standard deviations. * denotes a significant difference (P<0.05) compared with untreated cells.