A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis

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.

Figure 1. Protocol for preparation of cecal slurry (CS) stock for cryopreservation.

Cecal contents were aseptically collected from mice and prepared according to the diagram. An additional filtration step using a 70 µm strainer is recommended after step D.

Figure 2. Abnormal cecum characteristics in mice with diabetes, chronic pancreatitis, and aging.

Ceca and cecal contents were collected from mice under different experimental conditions. Wet weight of cecal contents (A), wet/dry weight ratio of cecal contents (B), and whole cecum weight (C) from STZ-induced diabetic mice and non-treated control mice (n = 5 each). Macroscopic images (D), wet weight of cecal contents (E), wet/dry weight ratio of cecal contents (F), and whole cecum weight (G) from mice with caerulein-induced chronic pancreatitis and saline-injected control mice (n = 5 each). Macroscopic images (H), wet weight of cecal contents (I), wet/dry weight ratio of cecal contents (J), and whole cecum weight (K) from young and aged mice.

Figure 3. Bacteria quantification of cecal contents from young and aged mice.

Cecal contents from young and aged mice were suspended in sterile water and spread onto agar plates without antibiotics, with aztreonam to select for Gram-positive bacteria, or with Penicillin G to select for the majority of Gram-negative bacteria. Data represent the mean ± standard deviation, n = 5 for each group.

Figure 4. Bacterial viability of cecal slurry (CS) following different storage conditions.

Cecal contents from mice were prepared in 15% glycerol and CS samples were stored at −80, −20, and 4°C. Colony formation unit (CFU) was compared one and six weeks later for all storage conditions (A) and up to six months later for samples stored at −80°C (B). Data represent the mean ± standard deviation.

Figure 5. Survival curves of mice after injection with cecal slurry (CS).

Young, middle-aged, and aged C57BL/6 mice were injected with either (A) 100 µL, (B) 150 µL, or (C) 200 µL CS and survival monitored for ten days.

Figure 6. Circulating bacteria amount correlates with mortality.

(A) Blood was taken from the tail vein of young, middle-aged, and aged mice 12, 24, or 48 h after injection with 150 µL CS and colony formation unit (CFU) assessed by spreading on agar plates. After fifteen days, data from mice which survived or died were separated into two groups and CFU compared. (B) Blood was taken from the tail vein of young mice 24 h after injection with 200 or 400 µL CS and CFU assessed by spreading on agar plates. After ten days, data from mice which survived or died were separated into two groups and CFU compared. Each symbol represents data from a single animal.

Figure 7. Heat-inactivation of cecal slurry (CS) induces endotoxemia but does not cause mortality after injection to mice.

Mice (13-month-old) were injected with 200 µL of untreated CS (in 15% glycerol/PBS), heat-inactivated (72°C, 15 min) CS, or vehicle (15% glycerol) and the body temperatures monitored for several days. CS injected mice died within 48 hours, while no mortality was observed in other groups. Data represent the mean ± standard deviation, n = 3 for each group).