Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery

Foxp3+Foxp3^+ Regulatory T Cells Are Required for Recovery from Severe Sepsis

The role of regulatory T cells (Tregs) in bacterial sepsis remains controversial because antibody-mediated depletion experiments gave conflicting results. We employed DEREG mice (DEpletion of REGulatory T cells) and a caecal ligation and puncture model to elucidate the role of $CD4^+Foxp3^+$ Tregs in sepsis. In DEREG mice natural Tregs can be visualized easily and selectively depleted by diphtheria toxin because the animals express the diphtheria toxin receptor and enhanced green fluorescent protein as a fusion protein under the control of the foxp3 locus. We confirmed rapid Treg-activation and an increased ratio of Tregs to Teffs in sepsis. Nevertheless, 24 h after sepsis induction, Treg-depleted and control mice showed equally strong inflammation, immune cell immigration into the peritoneum and bacterial dissemination. During the first 36 h of disease survival was not influenced by Treg-depletion. Later, however, only Treg-competent animals recovered from the insult. We conclude that the suppressive capacity of Tregs is not sufficient to control overwhelming inflammation and early mortality, but is a prerequisite for the recovery from severe sepsis

The Role of the Vagus Nerve: Modulation of the Inflammatory Reaction in Murine Polymicrobial Sepsis

The particular importance of the vagus nerve for the pathophysiology of peritonitis becomes more and more apparent. In this work we provide evidence for the vagal modulation of inflammation in the murine model of colon ascendens stent peritonitis (CASP). Vagotomy significantly increases mortality in polymicrobial sepsis. This effect is not accounted for by the dilatation of gastric volume following vagotomy. As the stimulation of cholinergic receptors by nicotine has no therapeutic effect, the lack of nicotine is also not the reason for the reduced survival rate. In fact, increased septic mortality is a consequence of the absent modulating influence of the vagus nerve on the immune system: we detected significantly elevated serum corticosterone levels in vagotomised mice 24 h following CASP and a decreased ex vivo TNF-alpha secretion of Kupffer cells upon stimulation with LPS. In conclusion, the vagus nerve has a modulating influence in polymicrobial sepsis by attenuating the immune dysregulation

Serum cytokines in CLP-treated mice.

<p>DEREG and C57BL/6 mice pre-treated with DT (1 µg in 100 µl PBS i.v. on days −2 and −1) were subjected to CLP or left untreated. Twenty-four hours later serum cytokine concentrations (IL6, IL10, MCP-1, IFNγ, TNFα and IL12p70) increased in septic animals, but there were no differences between Treg-depleted and non-depleted septic mice. Means are indicated; n = 5–9 mice/group.</p

Depletion of Tregs did not alter cell migration into the peritoneum.

<p>DEREG and C57BL/6 mice pre-treated with DT (1 µg in 100 µl PBS i.v. on days −2 and −1) were subjected to CLP (n = 7–9 mice/group) or left untreated (n = 5 mice/group). Cells from the peritoneal cavity were harvested by peritoneal lavage 24 h after CLP. The total number of cells was enumerated and related to volume of recovered lavage fluid (A). The numbers of B cells (CD19⁺) (B), neutrophils (Ly6G⁺) (C), peritoneal macrophages (F4/80⁺CD11b⁺MHCII⁺) (D) and inflammatory monocytes (F4/80^-CD11b⁺MHCII^lo) (E) was assessed via flow cytometry. Means are shown.</p

Treg-depletion augmented disease severity and decreased survival in CLP-treated mice.

<p>DEREG and C57BL/6 mice pre-treated with DT (1 µg in 100 µl PBS i.v. on days −2 and −1) were subjected to CLP. Survival was monitored for 5 days (A) and disease severity was scored for 3 days (B) by an observer blinded for the group assignment. Data from four independent experiments with similar results are summarized (each experiment involved 8–11 mice/group). Median and interquartile range of the disease severity score are shown in panel B; ** p<0.01.</p