Evaluation of urination in a rat cystitis model of inducedby hydrogen peroxide○ Hiromu Shimizu, Emi Morita, Saeko Yoshihara, Daichi Makabe, Ryoji Mizumachi,Takashi Tashiro, Seiichi Katayama, Naoyuki Hironaka, Katsuhide Nishi

Summary in Japanese

Objective

排尿障害治療のため，各種の動物モデルおよび評価方法が用いられているが，長 期間の評価が 可能な膀胱 炎モデルは少ない．我々が 現有する酢酸，塩酸，シクロホスファミド誘発膀胱炎モデルでは，いずれも頻尿症状が 1 週間ほどで回復する．そこで今回，過酸化水素（H2O2）を用いて，長期に頻尿症状が継続する膀胱炎モデルの構築を行った．
雌性 SDラットを用い，まず H2O2 による膀胱炎の惹起時間の検討と病態モデルの維持期間を確認した．イソフルラン吸入麻酔下で 3% H2O2 を膀胱内に注入し，5 分，15 分，30 分貯留する群を設け，その後 1，2，3，4, 5 週でウロフロメトリー測 定を行った．次に Amitriptyline，Celecoxib を用いて薬 剤 効 果検証を行った．
膀 胱 内 H2O2 貯 留 5 分では 排 尿 間 隔の 短 縮 がみられたが 個 体差が大きく，30 分では病態モデル作製 3 週後までに動物が死亡したため，以後の実験には15 分を用いた．この条件でウロフロメトリー測定では病態モデル作製 2 週後まで排尿間隔の短縮が認められた．
薬効評価では Amitriptyline（間質性膀胱炎・膀胱痛症候群診療ガイドラインで推奨グレードB）で頻尿症状の改善が認められたが，膀胱痛を抑えることで排 尿 機 能 を 改 善 する可 能 性 の あ るCox-2 阻 害 薬 であ るCelecoxibで は明らかな変化は見られなかった．
このモデルは現行モデルより1 週間長く膀胱炎評価が可能であるため，膀胱炎治療薬の薬効評価に有用であると考えられた．

Among various interstitial cystitis models in animals, few can be suitable for a long term evaluation. Although wehave acetic acid-, hydrochloric acid-, and cyclophosphamide-induced models, the symptoms of pollakiuria recover
within a week. Therefore, we tried to establish a cystitis model in which frequent urination symptoms continue fora long period of time using hydrogen peroxide (HP).
Female SD rats were used in the present study. First, we tested the induction time of HP and the maintenance periodof symptoms. The model rats were prepared by injection of 3% HP into the bladder under isoﬂurane inhalation
anesthesia. HP was stored in the bladder for 5, 15, or 30 min for separate groups. Uroﬂowmetry was measured at 1, 2,3, 4, and 5 weeks after model preparation.
As the results, HP stored for 5 min shortened the urination interval, but individual diﬀerences were large. All rats diedfor 30 min condition. Therefore, we used 15 min thereafter. In this condition, the micturition interval was found to beshorten up to 2 weeks. Next, we tested the eﬀects of amitriptyline (Grade B: Clinical Guideline for Interstitial
Cystitis/Bladder Pain Syndrome) and celecoxib (Cox-2 inhibiting NSAID). Although amitriptyline improved the frequenturination symptoms, celecoxib did not show the improvement. As the conclusion, the HP-induced cystitis model isthought to be useful for evaluation of cystitis remedy because we could extend the observation period one weeklonger than the currently available models.

Materials and Methods
Experiment 1: Examination of model production conditions

Experiment 2: Evaluation of drug eﬃcacy in an H2O2-induced cystitis model● Animals● Animals● Procedure● Procedure

Female rat, Crl:CD(SD), 9-week-old at model preparation

Female rat, Crl:CD(SD), 9-week-old at model preparation

1) The model was prepared with an intravesical retention time of 15 minutes under the conditions ofExperiment 1.
2) The dosing solutions were administrated orally once a day for 3 weeks from the day after model
preparation.
3) Uroﬂowmetry was measured at 1, 2, and 3 weeks after model preparation.

1) Under 2% isoﬂurane anesthesia, a polyethylene tube was introduced into thebladder transurethrally and then the lower abdomen was pressed gently towithdraw urine.
2) 300 µL of 3% H2O2 solution dissolved in sterile saline was introduced into thebladder through the catheter.
3) H2O2 in bladder was suctioned 5, 15, or 30 minutes after injection.
4) Uroﬂowmetry was measured at 1, 2, 3, 4, and 5 weeks after model preparation.
Test group

Dosing volume(mL/body)

3% H2O2NNormal--

3

5 min

0.3

5 min

315 min0.3

15 min

330 min

0.3

30 min3Time Schedule：Uroﬂowmetry

Model Preparation

Test group
NormalSaline

0-5Control

Saline0+51+510+

5Amitriptyline Amitriptyline

Week 01234

Celecoxib

5Time Schedule

Dosing volume 3% H O
2 2 n(mg/kg)

Dosingsolution

Celecoxib：Uroﬂowmetry

Model Preparation

Week 012

Administration

3

Results
Experiment 1

Experiment 2

Fig. 1(a)Fig. 2(b)Voiding interval (s)

9000
8000

1.2

Normal7000

Bladder weight at 2w

Voiding volume (g)1.4Normal

4000

*3000

*

2000
1000

**

00w

5 min********30 min

1w

0.62w

0.4

15 min3w

Model Preparation

4w

0.2

5w

0.0(g/100 g B.W.)

0.06

**(a)

5 min

*

*

15 min
*0w

2000

0.02

*

30 min

**

2w

3w

1w

4w

Amitriptyline
Celecoxib
NormalControl

Celecoxib
NormalAmitriptyline
Control

#
**
**

1000

**

0.6
#

0.4

*
*

0.2

0.01

5w

0.00

0NormalH2O2-15 min

0w

1W

2W

0.0

3W

Model Preparation

Model Preparation(ｃ)

In 30 min group, all animals were dead by the time of the 3w measurement
Each value represents the mean ± S.E. (n＝3)*: p<0.05, **: p<0.01; Signiﬁcant diﬀerence from Normal group (Studentʼs -test)(ｄ)Qmax (g/s)

0.6

Photo 1

NormalAmitriptyline
Control
Celecoxib0.3(c)
UrotheliumInterstitial
Smoothmuscle layer

0.4

3W

0.1

0.1

0.0

0.0

2W

3W

Model Preparation

NormalAmitriptyline
Control
Celecoxib

0.3
0.2

1W

2W

0.5

0.2

0w

1W

Qave (g/s)

0.6

0.4(b)

0w

Model Preparation

0.5

(a)

Voiding volume (g)

0.8

4000

30000.03(b)Voiding interval (s)

0.04

0.8

5000

0.07

0.05

1.06000

Fig. 30w

1W

2W

3W

Model Preparation

Qmax: maximal urinary ﬂow rate
Qave: average urinary ﬂow rate
Each value represents the mean ± S.E. (n＝5)*: p<0.05, **: p<0.01; Signiﬁcant diﬀerence from Normal group (Student's t-test)#: p<0.05; Signiﬁcant diﬀerence from Control group (Student's t-test)

Experiment 1Discussion

・ When H2O2 was stored in the bladder for 5 min, the voiding interval was shortened, though there were large individual diﬀerences. H2O2 for 30 min was mortal [Fig. 1-(a)].
・ In the H2O2 -induced cystitis model, frequent urination lasted for up to 2 weeks and then gradually recovered [Fig. 1-(a),(b)].
・ Uroepithelial hyperplasia, submucosal inﬂammatory cell inﬁltration and edema, and hemorrhage were observed in a 2-week model [Photo 1-(b),(c)].
・ Bladder weight increased after 2 weeks [Fig. 2].

Since these ﬁndings similar to that found in humans, storage of H2O2 in the bladder for 15 min could be a valid model.

Experiment 2・ Amitriptyline improved frequent urination [Fig. 3-(a),(b)].
・ No clear changes were observed by celecoxib [Fig. 3-(a),(d)].

The 3% H2O2 -induced cystitis model showed longer-lasting symptoms. Moreover, the current histopathological ﬁndings were not obtained in our previous models.
Since long-term evaluation of treatments could be observable, the current model is thought to be useful for evaluation of cystitis remedy.