Fig 1.
Experimental design of the study.
In a first experimental setup (A), mice received anti-interleukin-6 antibodies (1 mg/kg) or the vehicle (IgG1 kappa isotype) simultaneously with the CLP- or sham-procedure. In a second setup (B), mice furthermore received a repeated injection with the antibodies 24h following the procedure. In a third setup (C), mice only received anti-IL-6 or vehicle 24h following the CLP- or sham-procedure. 48h post-CLP, mice received a gavage of colored solid beads and 2h later mice were sacrificed for measurement gastrointestinal transit, prelevation of serum and tissue samples, measurement of colonic permeability and quantification of cell adhesion molecules at the mRNA and protein level. CBA: cytometric bead array; CDS: clinical disease score; CLP: caecal ligation and puncture; MLN: mesenteric lymph nodes; mRNA: messenger ribonucleic acid; RT-PCR: reverse transcriptase real-time polymerase chain reaction.
Table 1.
The clinical disease score (CDS) as applied in our study to score signs of disease in individual mice.
Fig 2.
Effect of preventive treatment with anti-IL-6 antibodies on sepsis-induced clinical signs of disease 24h (A) and 48h (B) following CLP or sham-surgery, percentage of weight loss at day 1 (C), percentage of gastric emptying (D), geometric center of GI transit (E) and colonic permeability as measured by the Evans blue method (F). Two-way ANOVA followed by One-way ANOVA and SNK post-hoc testing when appropriate, or its non-parametric equivalent for ordinal data; n = 7–10/group for A, B and C; n = 9–12/group for D; *p ≤ 0.05, ***p ≤ 0/001, # significant effect of CLP, § significant effect of anti-IL-6. CDS: clinical disease score; CLP: caecal ligation and puncture; GC: geometric center; %GE: percentage of gastric emptying; GI: gastrointestinal.
Table 2.
Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA or ELISA (IL-1α), and determined by RT-PCR in colon (C) during the preventive set-up (administration of anti-IL-6 antibodies simultaneously with the CLP- or sham-procedure).
Table 3.
Cultures of blood and homogenized mesenteric lymph nodes.
Table 4.
RT-PCR of cell adhesion molecules.
Fig 3.
Effect of preventive treatment and repeat injection with anti-IL-6 antibodies on sepsis-induced clinical signs of disease 24h (A) and 48 h (B) following CLP or sham-surgery, percentage of weight loss at day 1 (C), percentage of gastric emptying (D), geometric center of GI transit (E) and colonic permeability as measured by the Evans blue technique (F). Two-way ANOVA followed by One-way ANOVA and SNK post-hoc testing when appropriate, or its non-parametric equivalent for ordinal data; n = 7–10/group for A, B and C; n = 9–12/group for D; *p ≤ 0.05, # significant effect of CLP, § significant effect of anti-IL-6. CDS: clinical disease score; CLP: caecal ligation and puncture; GC: geometric center; %GE: percentage of gastric emptying; GI: gastrointestinal.
Fig 4.
Kaplan-Meier survival analysis.
Kaplan-Meier curve displaying survival of sham and CLP-mice treated with anti-IL-6 antibodies or IgG isotype control up until 48h following the CLP-procedure. Log rank test p = 0.011.
Table 5.
Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA, and determined by RT-PCR in colon (C) during the preventive set-up with repeated injection (administration of anti-IL-6 antibodies simultaneously with the CLP- or sham-procedure, and repeated once 24h following the procedure).
Fig 5.
Effect of curative treatment with anti-IL-6 antibodies on sepsis-induced clinical signs of disease 24h (A) and 48h (B) following CLP or sham-surgery, percentage of weight loss at day 1 (C), percentage of gastric emptying (D), geometric center of GI transit (E) and colonic permeability as measured by the Evans blue method (F). Two-way ANOVA followed by One-way ANOVA and SNK post-hoc testing when appropriate, or its non-parametric equivalent for ordinal data; n = 7–10/group for A, B and C; n = 9–12/group for D; *p ≤ 0.05, # significant effect of CLP. CDS: clinical disease score; CLP: caecal ligation and puncture; GC: geometric center; %GE: percentage of gastric emptying; GI: gastrointestinal.
Table 6.
Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA, and determined by RT-PCR in colon (C) during the curative set-up (administration of anti-IL-6 antibodies 24h following the CLP- or sham-procedure).