Abstract
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.
Highlights
Sepsis is a life-threatening clinical condition characterized by a profound systemic inflammatory response to infection
The severity of cecal ligation and puncture (CLP)-induced sepsis is highly dependent on the degree of infection which can be influenced by volume, rapidity, and duration of cecal content released into the abdomen, and by the bacterial flora present in the cecum
Cecal content wet weight were not different between the two groups (Fig. 2A), but cecal content wet/dry ratio (Fig. 2B) showed a significant difference indicating that fecal matter within the cecum of diabetic mice has a different consistency than normal mice
Summary
Sepsis is a life-threatening clinical condition characterized by a profound systemic inflammatory response to infection. Sepsis affects more than 700,000 people and claims at least 200,000 lives in the US annually [1] This disease is serious among the elderly population as both incidence and mortality drastically increase with advanced age [2]. The severity of CLP-induced sepsis is highly dependent on the degree of infection which can be influenced by volume, rapidity, and duration of cecal content released into the abdomen, and by the bacterial flora present in the cecum. Despite its popularity, CLP may not be preferable for certain experiments including those investigating animals with different cecum size, shape, or bacterial flora. This concern may apply for studies that compare severity of sepsis among animals with different body size This concern may apply for studies that compare severity of sepsis among animals with different body size (ex. neonatal mice or mutant dwarf mice), under different diet regimens (ex. liquid diet, high fat diet, or diet restriction), different gastrointestinal conditions (ex. neonatal or aged animals or animals with gastrointestinal pathology), or increased sensitivity to surgery (ex. aged animals or animals with deficient wound-healing capability)
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