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).