Table 1.
Histological injury score (HIS).
Table 2.
Histological assessment of colitis parameters (HAC).
Table 3.
Primer sequences used for quantitative RT-PCR.
Fig 1.
Characterization of oxazolone-induced colitis model.
(A) Wild type BALB/c mice were sensitized by epicutaneous application of 3% oxazolone at a dilution of 4:1 in a mixture of acetone and olive oil (100μl) on day 0, followed by intracolonic administration of 1% oxazolone dissolved in 50% ethanol (100μl) (or 50% ethanol for control group EtOH-treated mice) on day 7 using an intravenous catheter inserted 3 cm in the colon. Mice were sacrificed 6, 24 hours and 5 days post oxazolone administration and tissue samples were collected for different analysis. (B) Survival proportion was assessed daily. Mortality is expressed as survival rate and shown by Kaplan-Meier survival curves; statistical significance of Kaplan-Meier survival curves was determined with Gehan-Breslow-Wilcoxon test. *p < 0.05; (n = 12–18 mice per group). (C) Repeated measurements of body temperature of EtOH- and oxazolone-treated mice were taken every 30 minutes and followed until 6 hours after treatment with oxazolone. Body temperature is shown as mean ± SEM, as determined by the repeated-measures two-way ANOVA test. ****p < 0.001; (n = 8 mice per group). (D) Bar graphs represent serum levels of HMGB1 in naïve, EtOH- and oxazolone-treated mice 6h post colitis induction as determined by the Mann-Whitney test. Ns, not significant; (n = 3–4 mice per group). (E) Paraffin-embedded colon sections were stained with hematoxylin and eosin (H&E) assessment of tissue alteration. Representative images of colonic sections stained with H&E from control naïve, EtOH- and oxazolone-treated mice at 6 and 24 hours after colitis induction. Scale bars are 40 μm in the upper panel and 100 μm in the lower panel. (F) Th1, chemokines and Th2 (G) gene expression was quantified in colonic tissue isolated from EtOH- and oxazolone-treated mice. Data are expressed as mean ± SEM of fold increase versus naïve mice as determined by the repeated-measures two-way ANOVA test. Ns, not significant; *p < 0.05; (n = 3–6 mice per group).
Fig 2.
Immune profile of oxazolone-induced colitis.
(A) Representative expression of DAPI and CD11b (left column), Ly6G and CD11b (mid-left column), SiglecF and CD11b (mid-right column) and Ly6C and MHCII (right column) from the colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Frequencies (B) and absolute numbers (C) of Ly6G+CD11b+, Ly6C+MHCII- and Ly6C-MHCII+ and SiglecF+CD11b+ cells among CD11b+, SiglecF-CD11b+, Ly6G-CD11b+ cells from colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Data are expressed as mean ± SEM as determined by the Mann-Whitney test. ns, not significant; *p < 0.05; (n = 3–6 mice per group).
Fig 3.
Induction of Th2 and NK T cells is specific to oxazolone-induced colitis treatment.
(A) Representative expression of DAPI-CD45+ cells (left column), CD49b+ and FceRIa+ cells (mid-left column), CD3+CD45+ cells (mid-right column) and Ly49c+CD3+ cells (right column), and DAPI-CD45+ cells (left column) and CD4+IL5+ cells (right column) from the colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Frequencies (B) and absolute numbers (C) of CD49b+ FceRIa+, CD3+CD49c+, CD4+IL5+ cells from colon of naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction. Data are expressed as mean ± SEM as determined by the Mann-Whitney test. Ns, not significant; *p < 0.05; **p < 0.005; (n = 3–6 mice per group).
Fig 4.
Oxazolone exposure leads to early induction of systemic cytokines.
(A-C) Bar graphs represent cytokines in serum and isolated splenocytes of oxazolone-treated mice in both early (6h) and late (24h) phases of the disease. Data are expressed as mean ± SEM as determined by the repeated-measures two-way ANOVA test. ns, not significant; *p < 0.05; (n = 4 mice per group). (D) TNFα and IL6 immunofluorescence on spleen and colon section of naïve and oxazolone-treated mice 6 hours post colitis induction. Scale bars are 40 μm.
Fig 5.
Vagal nerve stimulation improves survival in oxazolone-induced colitis.
(A) Wild type BALB/c mice were sensitized by epicutaneous application of 3% oxazolone at a dilution of 4:1 in a mixture of acetone and olive oil (100μl) on day 0, followed by VNS or sham treatment and intracolonic administration of 1% oxazolone dissolved in 50% ethanol (100μl) (or 50% ethanol for control group EtOH-treated mice) on day 7 using an intravenous catheter inserted 3 cm in the colon. Mice were sacrificed 6 hours and 5 days post oxazolone administration and tissue samples were collected for different analysis. (B) Survival proportion was assessed daily. Mortality is expressed as survival rate and shown by Kaplan-Meier survival curves; statistical significance of Kaplan-Meier survival curves was determined with Gehan-Breslow-Wilcoxon test. **p < 0.005; (n = 14–16 mice per group). (C) Repeated measurements of body temperature of EtOH- and oxazolone-treated mice were taken every 30 minutes and followed until 6 hours post colitis induction. Body temperature is shown as mean ± SEM, as determined by the repeated-measures two-way ANOVA test. ****p < 0.001; (n = 7–8 mice per group). (D) Bar graphs represent serum levels of HMGB1 in naïve, EtOH- and oxazolone-treated mice 6 hours post colitis induction as determined by the Mann-Whitney test. *p > 0.05; (n = 2–16 mice per group). (E) Paraffin-embedded colon sections were stained with hematoxylin and eosin (H&E) assessment of tissue alteration. Representative images of colonic sections stained with H&E from control sham and VNS-treated mice 6 hours post colitis induction. Scale bars are 40 μm in the upper panel and 100 μm in the lower panel. (F) Bar graphs represent histological injury score and histological assessment of colitis score of colonic samples of sham and VNS-treated mice 6 hours post colitis induction. Data are expressed as mean ± SEM as determined by the Mann-Whitney test. Ns, not significant; (n = 7–8 mice per group).
Fig 6.
Vagal nerve stimulation reduces colonic and systemic inflammation.
(A-B) Bar graphs represent Th1 (A) and Th2 (B) gene expression in colonic tissue isolated from sham- and VNS-treated mice challenged with oxazolone 6 hours after colitis induction. (C) Bar graphs represent serum levels of IL6, KC and TNFα in sham- and VNS-treated mice challenged with oxazolone 6 hours after colitis induction as determined by the Mann-Whitney test. Data are expressed as mean ± SEM; *p < 0.05; ns, not significant; (n = 21–27 mice per group).