Evaluation and comparison of two kinds of LPSinduced acute lung injury (ARI) mice models
2種類のLPS誘発急性肺炎症モデルの評価と比較〇Souta Ushijima, Sayuri Nishikawa, Hideaki Kamaga, Kousuke Morizumi, Seiichi Katayama, Katsuhide Nishi
LSIM Safety Institute Corporation, Kumamoto Laboratories, Pharmacology Department

・ Two kinds of LPS-induced acute lung injury mice models were confirmed that it able to evaluate.
① LPS inhalation model
Inflammatory cell infiltration in BALF was confirmed and dexamethasone (10 mg/kg) improved thisinfiltration.
② α-GalCer-LPS instillation modelA weight loss, the abnormality of the general state and lung inflammation after instillation of LPS wereconfirmed. Increase of the number of more remarkable cell infiltration in BALF was confirmed comparedto LPS inhalation model. There is no significant difference, this infiltration was seemed to be improvedby dexamethasone (30 mg/kg) under the study condition.
・ As a result, these two models were different in a characteristic each, and were able to use forⒸ 2021 LSIM Safety Institute Corporation All rights reservedevaluating
the drug.
1

COI開示☑ 発表内容に関連し、過去３年間、開示すべきCOI関係にある企業などはありません。
筆頭発表者: 牛島 壮太
責任発表者: 片山 誠一
2Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Introduction• Pneumonia is recognized as the serious condition of a patient by the expansion of
the new coronavirus (COVID-19) infectious disease. In addition, in the newcoronavirus infectious disease, the symptom of the acute pneumonia symptom totake a sudden turn is brought into problem in particular.
• LPS (lipopolysaccharide)–induced acute lung injury model is used for the study ofacute pneumonia symptom most, and we prepared and compared two mouse acutelung injury model using the LPS.
• One is a model to let LPS expose to a whole body, and another one is instillation ofLPS to nasal cavity under anesthesia with a pretreatment of α-garactosyl ceramid(αGalCer).
• These two animal model was evaluated by inflammation cell infiltration into BALF
and so on.
3Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Two LPS-induced acute lung injury model
Model 1
• LPS inhalation model
Reference: Natalia de Souza Xavier Costa et al. Early and late pulmonary effects ofnebulized LPS in mice An acute lung injury model. PLoS ONE 12(9):e0185474.

Model 2• α-GalCer-LPS instillation model
Reference: Hiroyasu Ito et al. Lethal endotoxic shock using α-galactosylceramide sensitization
as a new experimental model of septic shock. Laboratory Investigation (2006) 86,254–261
Tumurkhuu G. et al. The mechanism of development of acute lung injury in lethal
endotoxic shock using α-galactosylceramide sensitization. British Society forImmunology, Clinical and Experimental Immunology, (2008) 152: 182–191
4Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedTest System〇AnimalMice: BALB/cAnNCrlCrlj (Charles River, Japan)
Sex: ♂
Age: 6 weeks old (at purchased)〇Reagent
LPS: Lipopolysaccharides, from Escherichia coli O111:B4, Sigma-AldrichInc.
α-GalCer (α-galactosylceramide): Kirin, specific NKT activator
Dexamethasone: Sigma-Aldrich Inc.

5Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 1:LPS inhalation model＜Group configuration＞
GroupLPS

DoseN

Sham or Normal--LPS Inhalation

1 mg/mL, 30min-

3
3～4

LPS InhalationDexamethasone

1 mg/mL, 30min

10 mg/kg

3＜Schedule＞
Day 0-30分

6Day 10Dexamethasone LPS Inhalation
10 mg/kg，p.o.
30 min.

After about 24 hours

Serum, BALF,
Lung collectionⒸ 2021 LSIM Safety Institute Corporation All rights reservedModel 1:LPS inhalation model
8000

Total Cell

8000

**

6000

Cells / μL

6000
4000

##

27

Neutrophil

25

**

23
214000

2000

2000

00
LPS inhalation##

191715LPS inhalation

Sham

Body Weight (g)LPS inhalation

Day 0Day 1

Dexamethasone**: p<0.01, Significant difference from Sham group (Student t-test)##: p<0.01, Significant difference from LPS inhalation group (Student t-test)

Inflammatory cell infiltration in BALF, mainly neutrophil, induced by LPS inhalation wasconfirmed, and the change was improved by administration of dexamethasone.
7Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 1:LPS inhalation model (reproducibility)
Body Weight (g)16000

Cells / μL

14000

16000

Total Cell **27

Neutrophil

14000

25

12000

12000

10000

10000

8000

8000

21

6000

19

6000
4000

##

**

4000

2000

2000

0023

##

1715

LPS inhalation

LPS inhalation

NormalLPS inhalation

Day 0Day 1

Dexamethasone**: p<0.01, Significant difference from Normal group (Student t-test)##: p<0.01, Significant difference from LPS inhalation group (Student t-test)

The reproducibility of lung injury induced by LPS Inhalation was confirmed, but the differences
between experiments were existed.
8Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model＜Group configuration＞
Group

α-GalCer

LPS

DoseN

Normal---3α-GalCer-LPS

1 μg

5 μg

-

4

α-GalCer-LPSDexamethasone

1 μg

5 μg

10 mg/kg

3＜Schedule＞
Day 0α-GalCer
Instillation*9Day 3Day 1-30 min.

0

After about 48 hours

Dexamethasone
10 mg/kg, p.o.

LPS
Instillation*

Serum, BALF, Lung collection*: Administration into lung by instillation to nasal cavity under isoflurane inhalationⒸ 2021
anesthesia.
LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model
60000

Cells / μL

50000

Total Cell60000**

27

Neutrophil

2550000

**

40000

40000

30000

30000

2120000

20000

1910000

10000

1700

23

Normal****15
α-GalCer-LPS
instillation

α-GalCer-LPS
instillation

Body Weight (g)α-GalCer-LPSDay 0 Day 1 Day 2 Day 3

Dexamethasone**: p<0.01, Significant difference from Normal group (Student t-test)

Increase of the number of more remarkable cell infiltration in BALF was confirmed comparedto LPS inhalation model. Furthermore, dexamethasone was not able to improve the increase.
A weight loss, the abnormality of the general state and lung inflammation were confirmed.

10Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model (reproducibility)＜Group configuration＞
Group

α-GalCer

ShamPBSα-GalCer

1 μg

LPSPBSLPSDose(10 mL/kg)

3Saline
5 μg

Salline, p.o.

Model (α-GalCer-LPS )Dexamethasone

11N33
31 μg

5 μg

10 mg/kg, p.o.

330 mg/kg, p.o.

3Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model (reproducibility)
100000**Total Cell

Body Weight (g)30Cells / μL

80000

**

**

60000

2540000
20000

**

Sham

**

α-GalCer
LPS

20*

Model (α-GalCer-LPS)

0α-GalCer-LPS instillation
Sham

α-GalCerLPS

Model (α-GalCer-LPS)

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg15Day 0Day 1Day 2Day 3*: p<0.05, **: p<0.01, Significant difference from Sham group(Student t-test)

Inflammatory cell infiltration by only LPS instillation was confirmed sufficiently. However, by theinstillation of α-GalCer, the infiltration was stronger compared with only LPS instillation. There isno significant difference, this infiltration was seemed to be improved by administration of12dexamethasone(30 mg/kg).
Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model (oxygen saturation)
SpO2(%)

1051009590858075

α-GalCer-LPS instillation
Sham

α-GalCerLPS

Model (α-GalCer-LPS)

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

Reduction of SpO2 measured by pulse oximeter (Mouse OxPLUS, primetech) in Day 3 was confirmed
in LPS and model group. There is no significant difference, this reduction of SpO2 was seemed
to be improved by administration of dexamethasone.
13Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model (biochemical examination)
ALT (U/L)

20015015010010050500AST (U/L)

2000Shamα-GalCerSham

α-GalCer

LPS

Model (α-GalCer-LPS)

LPS

Model (α-GalCer-LPS)

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

ALP (U/L)
600

LDH (U/L)
1000

500

800

400

600

300

400

200

200

100014No strong systemic
inflammation wasconfirmed in this model
as a result of
measurement by
the dry chemistry.

0Sham

α-GalCer

Shamα-GalCer

LPS

Model (α-GalCer-LPS)

LPS

Model (α-GalCer-LPS)

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kgⒸ 2021 LSIM Safety Institute Corporation All rights reservedModel 2: α-GalCer-LPS instillation model (biochemical examination)
BUN (mg/dL)

Cre (mg/dL)
230
2011000Sham

α-GalCerSham

α-GalCerLPS

Model (α-GalCer-LPS)

LPS

Model (α-GalCer-LPS)

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

Dexamethasone 10 mg/kg

Dexamethasone 30 mg/kg

No renal disorder was confirmed in this model as a result of measurement by the dry chemistry.

15Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Summary・ Two kinds of LPS-induced acute lung injury mice models were confirmed that it able toevaluate.
① LPS inhalation model
Inflammatory cell infiltration in BALF was confirmed and this infiltration was improved byadministration of dexamethasone (10 mg/kg).
② α-GalCer-LPS instillation model
A weight loss, the abnormality of the general state and lung inflammation after instillationof LPS were confirmed. Increase of the number of more remarkable cell infiltration in BALF
was confirmed compared to LPS inhalation model. Furthermore, this infiltration was not
improve by dexamethasone (10 mg/kg). And there is no significant difference, this infiltration
was seemed to be improved by dexamethasone (30 mg/kg) under the study condition.
・ Both models were confirmed that there was the difference between experiments.
・ As a result, these two models were different in a characteristic each, and were able to use
for evaluating the drug.
・ About the cytokine storm, we are considering using BALF supernatant now.
16Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved