Study on the Evaluation System ofPost-Stroke Pain Model in Rats
〇Takashi Tashiro，Yoshiyuki Hayashida，Saeko Yoshihara，Masana Kamioka，〇Ikuo Takahashi，Seiichi Katayama，Naoyuki Hironaka，Katsuhide Nishi〇Kumamoto Laboratories, LSIM Safety Institute CorporationⒸ 2021 LSIM Safety Institute Corporation All rights reserved

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： 田代 貴士
責任発表者： 田代 貴士Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Objective
Central post-stroke pain (CPSP) is one of the most troublesome sequelae ofstroke. It is characterized by persistent pain, resulting in marked deteriorationin daily living activities and quality of life (QOL). In recent years, the rat
model of CPSP using collagenase-IV induced cerebral hemorrhage has been
increasingly reported.
In this study, we attempted to establish an assessment system of analgesics
in CPSP model. Male SD rats were induced cerebral hemorrhage by means ofmicroinjection of collagenase-IV into the lateral thalamic area. The pain
thresholds was evaluated with von Frey filament.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 1: Confirmation of the hemorrhagic lesion induced
by intra-thalamic collagenase【Animal】
Male rat, Crl:CD(SD), 8 weeks of age (at the model preparation)【Group configuration】
Group

Dose level(IU/head)

Dose volume(µL/head)

DosingrouteN

Model

0.125

0.5

i.c.

3

i.c.: intracerebral administration【Experiment schedule】
Model Preparation(Day 0)Confirmation of hemorrhagic lesion(Day 1)Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods【Animal】
Male rat, Crl:CD(SD), 8 weeks of age (at the model preparation)【Preparation of CPSP model 】(1) The animals were maintained under anesthesia with 2% isoflurane duringsurgery.
(2) The animals were injected with type IV collagenase (FUJIFILM Wako
Pure Chemical Corporation, 0.125 IU/0.5 µL saline) into the lateralthalamic area (anterior/posterior, -3.5 mm from bregma;lateral, 3.6 mm from the midline; ventral, 6.0 mm from the dura) using astereotaxic frame.
(3) Sham-operation animals were injected with 0.5 µL saline only.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reservedResults
Experiment 1: Confirmation of the hemorrhagic lesion induced
by intra-thalamic collagenase

Photos showing representative examples of the spatial extent of the hemorrhagic lesion induced
by intra-thalamic collagenase microinjection.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 2: Confirmation of the central post-stroke pain (CPSP)【Animal】
Male rat, Crl:CD(SD), 8 weeks of age (at the model preparation)【Group configuration】
Group

Dose level(IU/head)

Dose volume(µL/head)

DosingrouteN

Sham operation

00.5

i.c.

7

CPSP model

0.1250.5i.c.

5i.c.: intracerebral administration【Evaluation of the pain】
von Frey filament test
Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 2: Confirmation of the central post-stroke pain (CPSP)【Evaluation of the pain】
1Measurement of pain threshold with von frey filament
1Each of the filaments (0.4, 0.6, 1, 2, 4, 6, 8, 15, 26 g) was used according to1Chaplan's method.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 2: Confirmation of the central post-stroke pain (CPSP)【Experiment schedule】
Evaluationof the pain(Pre)

Modelpreparation(Day 0)Evaluation of the pain
Confirmation of(1W, 2W, 3W, 4W
hemorrhagic lesion
after the model preparation)(Day 29)Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Results
Experiment 2: Confirmation of the central post-stroke pain (CPSP)
Sham operation (n=7)

30

Right hindpaw

Pain threshold (g)Pain threshold (g)

30
25
20
1510

*

*

50
PreDay 7

Sham operation (n=7)

CPSP model (n=5)

Day 14

Day 21

Day 28CPSP model (n=5)

Left hindpaw

252015105**

Day 14Day 21*0Pre

Day 7Day 28

Mean ± S.E.
*: p<0.05, Significant difference from the sham operation group (Student's t -test).

Compared with the sham operation group, the CPSP model group showed lower pain thresholds fromDay 7 onwards, and a significant difference from Day 14 to Day 21 in both the left and the righthindpaws.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 3-1: Pharmacological study using CPSP model【Animal】
Male rat, Crl:CD(SD), 8 weeks of age (at the model preparation)【Group configuration】
GroupDosing
solutions

Dose level Dose volume(mg/kg)(mL/kg)DosingrouteN

Sham operation

saline02p.o.

5Vehiclesaline02p.o.

7

Pregabalinpregabalin

30

2p.o.

6i.c.: intracerebral administration【Evaluation of the pain】
von Frey filament test
Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 3-1: Pharmacological study using CPSP model【Experiment schedule】
2 weeks afterthe model preparation
Administration
Model
von Frey
preparation filament test
(Day 0)(Pre)

von Freyfilament test
(2, 4 hours
after administration)

Confirmation ofhemorrhagic lesionⒸ 2021 LSIM Safety Institute Corporation All rights reserved

Results
Experiment 3-1: Pharmacological study using CPSP model (CPSP)
30

Right hindpaw##Vehicle (n=7)##

25201510

**

**
**

5

Sham operation (n=5)
Pregabalin 30 mg/kg (n=6)

Pain threshold (g)Pain threshold (g）Sham operation (n=5)
Pregabalin 30 mg/kg (n=6)

30Vehicle (n=7)

Left hindpaw

##

##

**

**

**

Pre2h4h25
201510500Pre2h4h Mean ± S.E.

**: p<0.01, Significant difference from the sham operation group (Student's t -test).
##: p<0.01, Significant difference from the vehicle group (Student's t -test).

Compared with the sham operation group, the vehicle group showed a lower pain threshold, and asignificant difference was observed.
Compared with the vehicle group, the pregabalin group showed a higher pain threshold 2 hours and 4hours after administration, and a significant difference was observed in both left and right hindpaws.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and MethodsExperiment 3-2: Pharmacological study using CPSP model【Animal】
Male rat, Crl:CD(SD), 8 weeks of age (at the model preparation)【Group configuration】
GroupDosingsolutions

Dose level Dose volume(mg/kg)(mL/kg)DosingrouteN

Sham operation

saline02p.o.

7Vehiclesaline

02

p.o.

8

Pregabalinpregabalin

102p.o.

8i.c.: intracerebral administration【Evaluation of the pain】
von Frey filament test
Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Materials and Methods
Experiment 3-2: Pharmacological study using CPSP model【Experiment schedule】
2 weeks after
the model preparation
Administration
Modelvon Frey
preparation filament test
(Day 0)(Pre)

von Frey
filament test
(2, 4 hoursafter administration)

Confirmation ofhemorrhagic lesionⒸ 2021 LSIM Safety Institute Corporation All rights reserved

Results
Experiment 3-2: Pharmacological study using CPSP model (CPSP)
Sham operation (n=7)
Pregabalin 10 mg/kg (n=8)
2530

Right hindpaw##20##1510**Sham operation (n=7)
Pregabalin 10 mg/kg (n=8)

**5**

0

Pain threshold (g)Pain threshold (g)30Vehicle (n=8)

25Vehicle (n=8)

Left hindpaw
##20

##

1510**

**

**

Pre

2h4h50Pre2h4h Mean ± S.E.

**: p<0.01, Significant difference from the sham operation group (Student's t -test).
##: p<0.01, Significant difference from the vehicle group (Student's t -test).

Compared with the sham operation group, the vehicle group showed a lower pain threshold, and asignificant difference was observed.
Compared with the vehicle group, the pregabalin group showed a significantly higher pain thresholdadministration in both the left and the right hindpaws at 2 and 4 hours after administration.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved

Discussion
・ Male SD rats were used to prepare CPSP model rats by inducing cerebral
hemorrhage via a microinjection of collagenase-IV into the lateral thalamicarea.
・ Hemorrhagic lesions were observed in the lateral thalamic region of the・ CPSP model rats prepared by this method.
・ Pain was observed in the CPSP model rats compared with that in the sham・ operation rats. In addition, the analgesic efficacy of the pregabalin was・ confirmed in the CPSP model rats.
・ The results indicate that the CPSP model was established in rats.
・ Moreover, the CPSP model rats would be useful to evaluate potential
・ efficacy of newly introduced analgesics.

Ⓒ 2021 LSIM Safety Institute Corporation All rights reserved