Study on the establishment of a system
for measuring muscle strength of cisplatin-induced
muscle atrophy model in mice

○ Daichi Makabe，Emi Morita，Shinichi Ozato，Saeko Yoshihara，Hiromu Shimizu，Takashi Tashiro，Seiichi Katayama，Naoyuki Hironaka，Katsuhide Nishi

Objective

Summary in Japanese

抗がん剤の一種であるシスプラチンは，様々ながんの治療に広く使用されるが，重篤な副作用を伴うという問題点がある．その副作用の一つとして，骨格筋の萎縮が引き起こされることが，多数報告されている．筋委縮によるサルコペニアや，それに続く身体的フレイルは，患者のQOLを大きく低下させるため，薬の開発が必要である．また，薬の開発には筋委縮モデルにおける筋力低下を評価可能な試験系の確立が必要である．

Cisplatin is widely used in the treatment of various cancers, but it causes theatrophy of skeletal muscles. The atrophy might induce sarcopenia, which greatlyreduce the quality of life in patients. Therefore, it is necessary to establish an
evaluating test system of muscle weakness in muscle atrophy models.
Continuous periodic observations might be necessary to obtain precise informationon the effects of chemicals on muscle function. In literature, cisplatin-induced
muscle atrophy and improving effect of D-methionine in mice have been reported.
Therefore, we examined the possibility of periodic quantifying the effects of
D-methionine on cisplatin-induced muscle atrophy model by measuring muscle
strength using 1300A, 3-in-1 Whole Animal System (Aurora scientific).

Materials and Methods

本試験において，雄性C57BL/6J にシスプラチン（3 mg/kg/day, i.p.）を投与して筋委縮を惹起し，それに対して必須アミノ酸であるD-メチオニン（300 mg/kg/day,p.o.）を投与して効果を検証した．筋力はシスプラチンの初回投与より4日，７日および10日後に測定した．結果，作製したモデルの筋力は，正常マウスに比べ低下しており筋委縮が確認された．また，D-メチオニンが，筋委縮に伴う筋力低下を抑制することが確認された．この結果により，筋委縮モデルにおける筋力の経時的かつ定量的な測定を行う試験系を確立することができた．

● Schedule● AnimalMale mice, C57BL/6J, 8-week-old● Model preparation
Cisplatin was administrated by i.p. at a dose of 3 mg/kg for 9 days, once a day.

Day -2 -1● D-methionine administration
D-methionine was administrated by p.o. at a dose of 300 mg/kg, once a day and started
3 days before the first cisplatin administration.
● Group Configuration
GroupCisplatin
Dose and Route

D-methionine
Dose and RoutenNormalControl
D-methionine

Saline, i.p.
3 mg/kg, i.p.
3 mg/kg, i.p.

Saline, p.o.
Saline, p.o.
300 mg/kg, p.o.

101010muscle force measuring012345678910D-methionine administration
Cisplatin administration

1300A 3-in-1 Whole Animal System (Aurora Scientific)● Muscle force measuring
In this study, the muscle force was measured by 1300A 3-in-1 Whole Animal System(Aurora Scientific).
The mice were anesthetized by isoflurane inhalation and placed in the thermostaticallycontrolled table.
The foot firmly fixed to the footplate on the motor shaft. The muscle force values wereobtained via electrical stimulation of the plantar flexor muscles through the tibial nerve andmeasured with the dynamometer. Measurements were conducted at intervals of 4, 7 and10 days after cisplatin administration.
● Measurement condition- Tetanus force:
100Hz, 0.35sec- Force frequency: 20, 40, 60, 80, 100, 150 and 200 Hz, 0.35 sec, 60 sec interval- Fatigue:
30 Hz, 0.35 sec, 2 sec interval, 120 sec duration (60 stimulations)● Gastrocnemius muscle mass
Sampled gastrocnemius muscle and measured mass at days 10 after cisplatin administration.

Results
FIg. A Body weight change rate (%)

FIg. B

110

Tetanus force (mN)

FIg. C Force frequency-Day 10 (mN)
350

350

100

#90

**80

##**

**

##

**

Normal
##

**70

##

**

##

**D-methionine

300

##300

##

*250

Control##**200**

Normal250

D-methionine

200

Control

150

60

##

**

**

##

**

####

**

**

NormalD-methionine
Control

100
150

50
40

1234

5678

Each value represents the ｍean±S.E. (n=10)**P<0.01 vs Normal (Student’s t-test)#P<0.05, ##P<0.01 vs Control (Student’s t-test)

910 [Day]

FIg. D Fatigue-Day 10 (% Initial Force)

501004710

Each value represents the ｍean±S.E. (n=10)*P<0.05, **P<0.01 vs Normal (Student’s t-test)##P<0.01 vs Control (Student’s t-test)

FIg. E[Day]

Gastrocnemius muscle mass (g)020Hz 40Hz 60Hz 80Hz 100Hz 120Hz 150Hz 200Hz

Each value represents the ｍean±S.E. (n=10)**P<0.01 vs Normal (Two-way ANOVA, Student’s t-test)##P<0.01 vs Control (Two-way ANOVA, Student’s t-test)

Photo 10.16

300

0.14250##0.12200Normal*150

D-methionine
n.s. Control

100Gross pathology ofgastrocnemius muscle**0.10
0.08
0.06
0.04

500**

16111621 26 31 36 41 46 51 56 60 [time]

Each value represents the ｍean±S.E. (n=10)*P<0.05, **P<0.01 vs Normal (Two-way repeated measures ANOVA, Student’s t-test)
n.s. , No significant difference vs Control (Two-way repeated measures ANOVA)

0.02
0NormalControl

D-methionine

Each value represents the ｍean±S.E. (n=10)**P<0.01 vs Normal (Student’s t-test)##P<0.01 vs Control (Student’s t-test)

1 cmSummary and Conclusion● In control group, significant decrease in body weight after cisplatin administration was observed. In contrast, thisdecrease was significantly suppressed compared with control group in D-methionine-treated group [Fig A].
● Plantar flexion contractile force on day 4, 7 and 10 were significantly decreased in control group compared withnormal group, and this decrease of muscle force was significantly suppressed compared with control group inD-methionine-treated group [Fig B, C].
● The fatigue data decline was faster in control group than normal group. While fatigue ability improved byapproximately 20% compared to the control group after the 36th stimulus in D-methionine-treated group,D-methionine did not significantly affect fatigue ability [Fig D].
● Gastrocnemius muscle mass was significantly reduced in control group compared with normal group, and the
reduction of the gastrocnemius muscle mass was significantly suppressed in D-methionine-treated groupcompared with control group [Fig E, Photo 1].
● From the above, the muscle strength of the cisplatin-induced muscle atrophy mice was lower than that of normal
mice, and D-methionine improved muscle weakness.

In conclusion,This measurement system was able todetect the decline in muscle force ofmuscle atrophy model with highsensitivity, and useful for evaluatingchanges in mouse muscle force
periodically.
The model would be useful to evaluate
potential efficacy of newly therapeuticchemicals on myofunction.