Protocol for a Sepsis Model Utilizing Fecal Suspension in Mice: Fecal Suspension Intraperitoneal Injection Model

Abstract

Background: Various animal models of sepsis have been developed to optimize sepsis treatment. However, therapeutic agents that were successful in animal models were rarely effective in human clinical trials. The cecal ligation and puncture (CLP) model is currently the gold standard for sepsis studies. However, its limitations include the high variability among researchers and the difficulty in comparing animals with different cecum shapes and sizes. In this study, we established a protocol for the creation of a simple and accessible sepsis rodent model using fecal suspensions that minimized differences in technical effects among researchers and individual differences in animals.

Methods: A mouse model of sepsis using fecal suspension intraperitoneal injection (FSI) was created using fresh stool excreted within 24 h. The collected fresh stool was dissolved in saline solution and filtered. The obtained fecal suspension was injected intraperitoneally into the mice. Moreover, fecal suspensions with different concentrations were prepared, and the survival rates were compared among the fecal suspensions for each concentration. To assess the validity of the FSI as a sepsis model, CLP and FSI with similar mortality rates were compared pathologically, physiologically, immunologically, and bacteriologically. Histopathological comparison was evaluated by hematoxylin-eosin and Gram staining of the parenchymal organs. Physiological evaluation was performed by comparing the respiratory rate, body temperature, and blood gas analysis results. Immunological assessment was performed using multiplex analysis. Bacteriological comparisons were performed by culturing ascites fluid.

Results: The FSI model increased mortality in proportion to the fecal suspension concentration. The mortality rate was reduced with antibiotic administration. In various comparative experiments conducted using the FSI and CLP models, both models showed findings consistent with sepsis. Furthermore, the FSI model showed less variability among the individuals in each test.

Conclusion: This is the first detailed and accurate report of a protocol for creating a sepsis model using fecal suspension. The FSI model is a minimally invasive and accessible sepsis rodent model. Its clinical validity as a sepsis model was proven via histological, physiological, microbiological, and immunological evaluation methods. The FSI model minimizes individual differences between mice and helps to conduct accurate studies after the onset of sepsis.

Keywords: cecal ligation and puncture; cytokine; inflammation; mice; peritonitis; sepsis.

FIGURE 1

Procedure for creating the fecal suspension intraperitoneal injection (FSI) model. (A) FSI model with thin fecal suspension. (a) Collect the required amount of feces from the cage. Spread enough paper matting to prevent urine from mixing with feces. Eliminate as much non-fecal waste as possible and be careful not to let water drip from the water bottle. (b) Weigh out 400 mg (450 mg for making 30 mg/mL) from the collected feces. (c) Weigh out 40, 20, 10, 15, or 8 mL of saline solution into a tray, depending on the concentration of fecal suspension to be prepared. (d) Putting feces on a Falcon^® 70 μm cell strainer. (e) Soak the feces in the saline solution through the filter and macerate it thoroughly. (f) Filter the feces using a Falcon^® 70 μm cell strainer and grind stick (we used a syringe pusher as a grind stick). (g) Grind the feces well until it becomes a paste (until the feces is no longer gritty). After filtration, drain the water from the paste feces well. (h) The obtained fecal suspension is impurity-free and can be aspirated without resistance with a 25-gauge needle. (i) Transfer the fecal suspension to a container that can be stirred. (j) Shake the tube well to mix sufficiently. Step (j) is performed once for each procedure (k). (k) Immediately after Step (j), aspirate 1 ml of fecal suspension with a syringe. (l) Administer 1 ml of fecal suspension intraperitoneally to the mice with a 25-gauge needle. (B) FSI model with thick fecal suspension. (a) Collect the required amount of feces from the cage. Spread enough paper matting to prevent urine from mixing with feces. Eliminate as much non-fecal waste as possible and be careful not to let water drip from the water bottle. (b) Weigh out 8,000 mg from the collected feces. (c) Weigh out 80, 60, 53, 40, or 27 mL of saline solution into a tray, depending on the concentration of fecal suspension to be prepared. (d) Putting feces on a tea strainer. (e) Soak the feces in the saline solution through a tea strainer and macerate it thoroughly. (f) Filter the feces using a tea strainer and grind stick (We used a syringe pusher as a grind stick). (g) Grind the feces well until it becomes a paste (until the feces is no longer gritty). (h) After rough filtration, drain the water from the paste feces well. (i) The obtained fecal suspension is full of impurities and still cannot be aspirated with a 25-gauge needle. (j) Pour the fecal suspension onto a Falcon^® 70 μm cell strainer placed over a 50 ml tube. (k) Use a grind stick when necessary to promote filtration. (l) If the filter is clogged, remove the residue as needed. (m) The obtained fecal suspension is impurity-free and can be aspirated without resistance with a 25-gauge needle. (n) Shake the tube well to mix sufficiently. Step (n) is performed once for each procedure (o). (o) Immediately after Step (n), aspirate 1 mL of fecal suspension with a syringe. (p) Administer 1 ml of fecal suspension intraperitoneally to the mice with a 25-gauge needle.

FIGURE 2

Survival rates of each model mouse. (A) CLP model. (B) FSI model of thin fecal suspension (without antimicrobial administration). (C) FSI model of thick fecal suspension (with antimicrobial administration).

FIGURE 3

Results of ascites culture of FSI and CLP models 20 h after surgery. Results, 24 h after the start of capneic incubation with Sheep Blood Agar (A) FSI model, (B) CLP model.

FIGURE 4

Histopathological findings 20 h after onset of peritonitis in FSI model, CLP model and control cases. (A) Microscopic findings of the abdominal cavity. (B) 2.5×(Objective lens) lung tissues. (C) 40×(Objective lens) lung tissues. (D) 2.5×(Objective lens) spleen tissues. (E) 40×(Objective lens) spleen tissues. (F) 2.5×(Objective lens) liver tissues. (G) 20×(Objective lens) liver tissues. (H) 40×(Objective lens) liver tissues. (I) 5×(Objective lens) kidney tissues. (J) 40×(Objective lens) kidney tissues.

FIGURE 5

Heatmap analysis of cytokine and chemokine measurements performed by multiplex analysis for FSI and CLP models. Blood was evaluated with samples taken 20 h after the onset of peritonitis. Heat map showing relative evaluation of cytokine and chemokine values. The maximum value of each variable is red, the minimum value is green, and the intermediate value corresponds to the gradation scale from red to green. G-CSF, Granulocyte-colony stimulating factor; GM-CSF, Granulocyte macrophage colony-stimulating factor; IFNγ, Interferon gamma; IL, Interleukin; IP, Interferon gamma-induced protein; CXCL, C-X-C motif chemokine ligand; KC, Keratinocyte-derived chemokines; MCP, Monocyte chemotactic protein; CCL, C-C motif chemokine; MIP, Macrophage inflammatory protein; RANTES, Regulated on activation, normal T cell expressed and secreted; TNF, tumor necrosis factor.