Evaluation of a polymicrobial sepsis mouse model
induced by fecal suspension intraperitoneal injection
as usefulness for clinical application.
○ Masateru Furuta, Souta Ushijima, Hajime Wada, Kousuke Morizumi,Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Experiment 2： The blood cytokine concentration andhistopathological analysis in intense FSI model

Objective

本モデルは，血中サイトカイン濃度の上昇が認められたことから，高サイトカイン血症を生じるヒトの敗血症と類似しており，敗血症治療薬の薬効評価に有用であると考えられた．

Experiment 1： Confirmation and evaluation ofmild FSI model● AnimalMale mouse, Slc:ICR, 7-weeks-old at model preparation

5555＊ Vehicle was administered to vehicle control group.

0 2hr

6hr

24hr

＊ Vehicle was administrated tovehicle control group.

IFN-γ (pg/mL)

3006h

153440280250
12 24 36 48 60 72 84 96
Time after the fecal suspension injection (h)No significant difference between drug
administration and vehicle control group(Log-Rank test)

Vehicle control
Vancomycin 10 mg/kg×281102448

Vancomycin 10 mg/kg
Vancomycin 10 mg/kg×202hr

6hr

24hr

General condition observation every 12 hours

TNF-α (pg/mL)

6h

100

IL-1β (pg/mL)

IL-2 (pg/mL)

100

75

75

505024hIL-4 (pg/mL)

10025250006h

80000

24h

IL-6 (pg/mL)

60000

024h

Normal6h

24h

Vehicle control

6h

10000

24h

IL-10 (pg/mL)

600

5000

400

2500

200

6h

Vancomycin 10 mg/kg

24h

20Prednisolone 20 mg/kg

3Spleen

1024

48

72

Ileum

96

Time after the fecal suspension injection (h)Each value represents mean+S.E. of animals,
number above the dot: number of surviving
animals

Prednisolone 0.1 mg/kg×2
Predonisolone 1 mg/kg×2

Vancomycin
Prednisolone

In the FSI group, the survival rate was decreased and theconcentrations of cytokines increased compared with thenormal group [Fig. 2 to 3]. In addition, the tingible bodymacrophage in the spleen and ileum, bacterial
infiltration, and also degeneration / necrosis in thepancreas were observed in histopathological analysis[Fig. 4]. Hypercytokinemia was caused by an increase incytokines. Based on these results, the present model wasconsidered to represent clinical signs of sepsis.
Vancomycin improved the survival rate in the FSI model[Fig. 2]. The improved survival rate by antibiotics wasconsistent with clinical results. However, vancomycin orprednisolone did not induce clear changes in cytokine
concentrations or histopathological findings [Fig. 3 to 4].
This would be due to no anti-inflammatory effects ofvancomycin, and the hypercytokinemia was too strong tosuppress.
The FSI model was considered to be useful for evaluating
the efficacy of drugs because the signs of this model
were similar to the human sepsis.

Vehicle control

Conclusion: Bacteria infiltratiion,: Necrotic focus,: Tingible body macrophage24h

IL-17A (pg/mL)

8007500

06h

Fig. 4 Histopathological analysis

496

Time after the fecal suspension injection (h)Each value represents mean+S.E. of animals,
number above the dot: number of surviving
animals

Mean±SE, n=4 or 5, p<0.05, **: p<0.01, Significant difference from Normal (Student's t-test)
The horizontal axis shows the time after fecal suspension injection.

472

**

20000

Body Weight (g)3120

9Fecal suspension Injection (i.p.)
Vancomycin (s.c.)Prednisolone (s.c.)Serum collection
Intra-abdominal organs collection

40000

4060

1010101010400**

537001020-20096hr

80012 24 36 48 60 72 84 96
Time after the fecal suspension injection (h)*: p<0.05，**: p<0.01, significant differencecompared to vehicle control group(Log-Rank test)

Normal

100

25300

200

0Survival rate (%)

08Fig. 3 Blood cytokine concentration

0General condition observation every 12 hours

Fig. 128N＊ Vehicle was administered to normal and vehicle control group.

**24hr209Vehicle control
Predonisolone 20 mg/kg×22*2*
22-106hr

343196hrAdministration time
Doseafter fecal suspension(mg/kg)injection (hr)NormalVehicle Control
Vancomycin
Prednisolone
Satellite

37

*

40

General condition observation every 12 hours

100

Fecal suspension Injection (i.p.)
Vancomycin (s.c.)Prednisolone (s.c.)

0 1hr

**

60

Fecal suspension Injection (i.p.)
Vancomycin (s.c.)Prednisolone (s.c.)

Body Weight (g)

4080

*

010×2
0.1×2
1×2

10101010**

1, 6, 24*1, 61, 24
1, 24

0
10
10×2
20×2

2, 6, 24*
22, 62, 24

Vehicle Control
Vancomycin
Vancomycin
Prednisolone

Survival rate (%)

100

**

Vehicle Control
Vancomycin
Prednisolone
Prednisolone(mg/kg)NFig. 2**

・PBS pH 7.4 (1×): Phosphate-Buffered Saline, Thermo Fisher Scientific.
・Vancomycin hydrochloride for injection: Okura Pharmaceutical Co., Ltd.
・Prednisolone sodium succinate for injection: Shionogi Pharma Co., Ltd.
N

Administration timeDose
after fecal suspension(mg/kg)injection (hr)*● Reagents

Dose

Test group

Test group(1) The cecal contents collected from animals were suspended in PBS
and filtered.
(2) Sepsis symptoms were induced by fecal suspension intraperitoneal
injection at a dose of 1.6 g/kg.
(3) General condition was observed every 12 hours. Body weight wasmeasured every day.
(4) Vancomycin or prednisolone was injected subcutaneously at
arranged time.

Test group

Experiment 2-1

Experiment 2-2● Methods

Administration timeafter fecal suspension
injection (hr)・ Equipment: Bio-Plex Multiplex Immunoassay System (Bio-Rad Laboratories, Inc.)・ Kit: Bio-Plex Pro mouse cytokine G1 23-Plex panel (Targets: Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17A, KC, MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES,TNF-α)**

その結果，病態群では正常群と比較して，生存率の大幅な低下，血中における各種サイトカイン濃度の上昇が認められた．また，病理組織学的検査では，脾臓と回腸でマクロファージによる白血球の貪食，膵臓で菌体浸潤と壊死巣が認められた．敗血症様の所見が確認されたこの病態に対し，バンコマイシン投与は，生存率の有意な改善を示した．

● ReagentsThe same as Experiment 1.
● Measurement System**敗血症（Sepsis）は，生体への細菌感染により免疫反応が過剰に活性化する全身性の炎症反応である．最終的には臓器機能不全へ陥り，高い死亡率を示す．本報告において，我々は糞便懸濁液の腹腔内投与により，手術操作をせずに均質な多菌性敗血症を誘導できるモデル（FSIモデル）を作製した．さらに，本モデルに対するバンコマイシン及びプレドニゾロンの作用を生存率，血中サイトカイン濃度，腹腔内臓器の病理組織学的検査により検討した．

(1) The model induction method was the same as Experiment 1 (modification: 2.4 g/kg fecal suspension).
(2) Vancomycin or prednisolone was injected subcutaneously at arranged time.
(3) General condition was observed every 12 hours. Body weight was measured every day. [Experiment 2-1]
(4) Serum was collected 6, 24 hours after fecal suspension injection. [Experiment 2-2]
(5) Liver, kidneys, spleen, small intestine and pancreas were collected 24 hours after fecal suspension
injection. [Experiment 2-2]**

Summary in Japanese● AnimalMale mouse, Slc:ICR, 7-week-old at model preparation● Methods**

Sepsis is caused by severe organ damage resulting from an
uncontrolled immune response by infection. In treatment ofsepsis in the early stage, antibiotics and other supportive therapy
are used. Animal sepsis models have been made mainly bycecal ligation and puncture (CLP) or lipopolysaccharide (LPS)
injection. However, these models are yet to be improved.
The objective of the present study was to induce the sepsis micemodel by fecal suspension intraperitoneal injection (FSI model)
and to evaluate validity of the model by administration ofvancomycin and prednisolone. We inspected the survival rate,
blood cytokine concentration, as well as histopathologicalanalysis of intra-abdominal organs.

Pancreas

6h24h