Figures & data
Figure 1. Representative graphs showing the cyclic oscillation of BL counts associated with active and inactive phases of intestinal inflammation induced by indomethacin administration. (A) Representative graph of BL count changes evolution in an indomethacin-treated rat and in a control rat. The cyclic oscillation of BL count was maintained for up to 60 days after drug administration. (B) Representative graph showing the timing of the start of the stress protocol. Stress exposure was performed during the inactive phase of inflammation following the second peak of high BL counts. The inactive phase was identified when two consecutive BL counts were less than the maximal BL count reached during the active phase. The gray zone of the graph corresponds to the 5 days of stress exposure using the protocol illustrated by the * stress scheme. WR, wrap restraint; WAS, water avoidance stress, each 1 h on alternate days.
Figure 2. Overview of the experimental design used in the study. Each indomethacin-treated rat was matched to a control rat. The stress/sham sessions started when the inactive phase following the second active phase of inflammation was identified in an indomethacin-treated rat. Simultaneously, the stress/sham sessions were started in their matched control individual. After 5 days of stress/sham exposure, both indomethacin and control rats were euthanized.
Table I. Primer sequences for RT-PCR.
Figure 3. Effects of indomethacin on percent of BW gain relative to the first day of drug administration. Data are mean ± SEM, n = 13–16 rats/group. A decrease in BW gain was observed after indomethacin administration (p < 0.001, ANOVA), whereas control rats showed a linear increase in this parameter.
Figure 4. Effects of stress on BW gain in control and indomethacin-treated rats. Data are mean ± SEM, n = 13–16 rats/group. (A) Time course of the percent weight gain relative to the first day of the stress/sham protocol in control and indomethacin-treated rats. (B) Percent of BW gain in control and indomethacin groups 5 days after stress exposure. Stress significantly decreased BW gain (two-way ANOVA, p = 0.0005). *p < 0.05 vs. control-nonstressed group; +p < 0.05 vs. indomethacin-nonstressed group.
Figure 5. Effect of stress exposure on fecal pellet output in control and indomethacin-treated rats. Date are mean ± SEM, n = 13–16 rats/group. Indomethacin-treated rats showed an increase in defecation frequency when compared with the healthy rats (two-way ANOVA, p = 0.004). Stress significantly increase pellet output when compared to the unstressed rats (two-way ANOVA p < 0.0001). ***p < 0.001 vs. control-nonstressed group: +++p < 0.001 vs. indomethacin nonstressed rats.
Figure 6. Effect of stress on BL counts in both control and indomethacin-treated rats. (A) Representative graph showing the time course of BL counts in an individual rat of each group. In each graph, the time that stress/sham exposure was initiated is indicated (rectangle in unstressed rats; oval in stressed rats). (B) Change in BL count 5 days after stress exposure expressed as percent of BL count recorded on the first day of the stress/sham protocol. Data are mean ± SEM, n = 13–16 animals/group. Stress significantly decreased BL levels (two-way ANOVA, p < 0.0001). This decrease was not related to the inflammatory state in indomethacin-treated rats (two-way ANOVA, p = 0.1125). ***p < 0.001 vs. control-nonstressed group; +++p < 0.001 vs. indomethacin-nonstressed group.
Table II. Number of animals with bacterial translocation and incidence for a specific micro-organism.
Figure 7. Effect of stress on the number of mucosal mast cells per VCU. (A–D): Microphotographs show RMCPII immunopositive cells (mucosal mast cells: dark staining) in the intestinal ileum mucosa of (A) control-nonstressed, (B) control-stressed, (C) indomethacin-nonstressed and (D) indomethacin-stressed animals. (E) Bar diagram representing the number of mucosal mast cells per VCU in ileum mucosa of each group. Three to five sections were counted per rat, 7–10 well-oriented VCUs were examined per section. Data are mean ± SEM, n = 13–16 rats/group. Indomethacin-treated rats showed an increase in mast cell count per VCU compared to the control group (two-way ANOVA, p = 0.0274). Stress also increased mast cell count per VCU both in control and indomethacin-treated rats (two-way ANOVA, p < 0.001). ***p < 0.001 vs. control-nonstressed group; +++p < 0.001 vs. indomethacin-nonstressed group.
Figure 8. Effect on iNOS mRNA expression in ileum of both control and indomethacin-treated rats. (A) Representative photograph of agarose gel showing RT-PCR products for inducible isoform of iNOS mRNA in ileum. C − , negative PCR control; CTRL (ns: control-nonstressed rat; s: control-stressed rat), and INDO (ns: indomethacin-nonstressed rat; s: indomethacin-stressed rat). (B) Bar diagram showing semiquantitative analysis by RT-PCR of iNOS mRNA expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Data are mean ± SEM, n = 13–16 animals/group. Stress induced a decrease in iNOS mRNA expression in control rats exposed to stressful stimuli. *p < 0.05 vs. control-nonstressed group, Student t-test.
