Sepsis, a life-threatening systemic inflammatory response syndrome induced by infection, is widely studied using laboratory animal models. While cecal-ligation and puncture (CLP) is considered the gold standard model for sepsis research, it may not be preferable for experiments comparing animals of different size or under different dietary regimens. By comparing cecum size, shape, and cecal content characteristics in mice under different experimental conditions (aging, diabetes, pancreatitis), we show that cecum variability could be problematic for some CLP experiments. The cecal slurry (CS) injection model, in which the cecal contents of a laboratory animal are injected intraperitoneally to other animals, is an alternative method for inducing polymicrobial sepsis; however, the CS must be freshly prepared under conventional protocols, which is a major disadvantage with respect to reproducibility and convenience. The objective of this study was to develop an improved CS preparation protocol that allows for long-term storage of CS with reproducible results. Using our new CS preparation protocol we found that bacterial viability is maintained for at least 6 months when the CS is prepared in 15% glycerol-PBS and stored at -80°C. To test sepsis-inducing efficacy of stored CS stocks, various amounts of CS were injected to young (4–6 months old), middle-aged (12–14 months old), and aged (24–26 months old) male C57BL/6 mice. Dose- and age-dependent mortality was observed with high reproducibility. Circulating bacteria levels strongly correlated with mortality suggesting an infection-mediated death. Further, injection with heat-inactivated CS resulted in acute hypothermia without mortality, indicating that CS-mediated death is not due to endotoxic shock. This new CS preparation protocol results in CS stocks which are durable for freezing preservation without loss of bacterial viability, allowing experiments to be performed more conveniently and with higher reproducibility than before.

Current animal models of sepsis often incorporate antibiotics to be consistent with clinical standards for treatment of patients in the ICU. However, such experimental intervention is commonly initiated very early after infectious insult, which likely blunts the progression of systemic inflammation and downstream pathology. The objective of this study was to establish an animal model of sepsis with delayed therapeutic intervention, allowing a longer disease course and downstream pathology, but still resulting in a high survival rate. Severe lethal abdominal infection was initiated in young adult (17-18 week-old) C57BL/6 mice by cecal slurry (CS) injection. When initiated early (1- or 6-hours post-CS injection), antibiotic treatment (imipenem, 1.5mg/mouse i.p., twice/day for 5 days) rescued the majority of mice; however, few of these mice showed evidence of bacteremia, cytokinemia, or organ injury. When antibiotic treatment was delayed until late time-points (12- or 24-hours post-CS injection) the majority of animals did not survive beyond 48 hours. When fluid resuscitation (physiological saline, s.c.) was performed in combination with antibiotic treatment (twice daily) beginning at these late time-points, the majority of mice survived (75%) and showed bacteremia, cytokinemia, organ dysfunction, and prolonged body weight loss (<90% for 4 weeks). We recommend that this new repeated combination treatment with antibiotics and fluids resuscitation be initiated at a late time point after bacteremia becomes evident because this model more closely mimics the downstream pathological characteristics of severe clinical sepsis yet maintains a high survival rate. This model would be advantageous for studies on severe sepsis and post intensive care illness.

Objective Visceral adipose tissue is a major site for expression of pro-inflammatory and pro-coagulant genes during acute systemic inflammation. In this study, we tested whether loss of fat mass by dietary restriction (DR) would remove the major source of these factors resulting in improved tolerance to sepsis and endotoxemia. Design Prospective, laboratory controlled experiments. Setting Aging and Critical Care Research Laboratory in a university hospital. Subjects Middle-aged (12 month-old) male C57BL/6 mice. Interventions Mice were subjected to 40% DR for three weeks followed by induction of abdominal sepsis or endotoxemia by intraperitoneal injection with cecal slurry or lipopolysaccharide (LPS), respectively. Measurements and main results Compared to freely-fed mice, DR mice exhibited dramatically improved survival (80% vs 0% after sepsis, p<0.001;86% vs 12%after endotoxemia, p=0.013) and significantly reduced visceral fat-derived mRNA expression of interleukin-6 (IL-6), thrombospondin-1, plasminogen activator inhibitor-1, and tissue factor which positively correlated with fat mass. Plasma levels of IL-6 were significantly reduced by DR and correlated with adipose IL-6 mRNAlevels and fat mass (p<0.001, R2=0.64 and 0.89). In vitro culture of visceral fat explants from naïve DR mice showed significantly reduced IL-6 secretion compared to that from freely-fed mice in response to LPS. Analysis of major adipose immune cell populations by flow cytometry demonstrated that macrophages were the only cell population reduced by DR and that CD11c+/CD206+ (M2-type) and CD11c-/CD206- (double negative) macrophages, in addition to T cells, are the major immune cell populations that produce IL-6 in middle-age during systemic inflammation. Conclusions Short-term DR drastically improved the survival outcome of middle-aged mice during both polymicrobial sepsis and sterile endotoxemia. Improved survival was accompanied by a significantly attenuated inflammatory response in adipose tissue, which is likely due to alterations of both fat mass quantity and qualitative changes including a reduction in macrophage populations.

No abstract

No abstract