Yonago Acta medica 2005;48:27–31

Effect of Long-Term Estrogen Replacement on Bladder
Function in Old Female Rats
Naoto Kobayashi, Katsuya Hikita and Ikuo Miyagawa
Division of Urology, Department of Surgery, School of Medicine, Tottori University Facultyof Medicine, Yonago 683-8504 JapanThe effects of estradiol (E2) on urodynamic parameters were studied with twenty
16-month-old female Wistar rats. They were divided into 4 groups, i.e., Group I: untreated; Groups II and III: treated with E2 for 4 and 8 weeks, respectively; Group IV:
treated with a placebo for 8 weeks. After treatment, we measured their plasma E2 levels,and recorded their voiding behavior for 24 h. Cystometry was performed and urodynamic parameters were analyzed. Particularly, bladder capacity as well as voided volume and frequency were surveyed. The results obtained were compared among groups.
Levels of bladder capacity in the E2-replaced groups (Groups II, 0.52 ± 0.14 mL andGroups III, 0.58 ± 0.09 mL) were signiﬁcantly (P < 0.05) higher than in the other groups(Group I, 0.38 ± 0.09 mL and Group IV, 0.40 ± 0.11 mL) respectively. The average voided volume was signiﬁcantly (P < 0.05) higher in the E2-replaced groups (Groups II, 1.06
± 0.22 mL and Groups III, 1.01 ± 0.16 mL) than in Group IV (0.79 ± 0.15 mL), respectively. Concerning the number of daily micturition per day, a signiﬁcant difference (P <0.05) was observed only between Group III (14.2 ± 2.7) and Group IV (18.8 ± 3.7). Thissuggests that E2-replacement therapy positively affects bladder function.
Key words: bladder function; estrogen; rat

pected to reverse the effects of urethral atrophy,
improving coaptation, and therefore increasing urethral closure pressure (Fantl et al., 1996). Furthermore, because estrogen has also been implicatedin raising the sensory threshold of the bladder tocholinergic stimulation, estrogen replacement therapy may be equally useful for detrusor instability(Ekstrom et al., 1993; Fantl et al., 1996).
To date, research on the success of estrogen
replacement therapy for postmenopausal bladderdysfunction has been saddled with controversy because many studies are based on subjective reports.
As early as 1941, Salmon et al. (1941) reported successful outcomes in postmenopausal women treatedwith intramuscular estrogen replacement therapy.

Urinary incontinence is a debilitating problem particularly prevalent in elderly women. Changes in
bladder function, associated with such postmenopausal states as detrusor instability, stress urinary
incontinence, and a propensity toward urinary tract
infection, are believed in part to be due to estrogendeﬁciency (Stenberg et al., 1995; Thom and Brown,1998). Lower urinary tract and pelvic floor tissues are known to be estrogen sensitive because
estrogen receptors are located throughout the bladder and urethra (Batra and Iosif, 1983; Klutke andBergman, 1995). For several reasons, it is hypothesized that estrogen replacement therapy may be
useful in treating urinary incontinence. In women
suffering from stress incontinence, estrogen is exAbbreviation: E2, estradiol.

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N. Kobayashi et al.
taken from the heart and was placed in a cyclone
separator to measure plasma E2 concentration levels.

Other investigators reported similar results (Rud,1980).
Animal studies have shed light on the role ofestrogen in bladder function. Longhurst et al. (1992)
found that estrogen treatment actually increases
detrusor sensitivity and improves bladder construction. We studied the urodynamic changes associated with estrogen depletion and replacement therapy.
In the current study we measured voiding behavior
and cystometry and assessed estrogen treatment.

Voiding behavior study
Each rat was placed in a metabolic cage containing
a urine collection funnel to record their voiding behavior. We designed these so that the urine moved
through a duct downward to a 250-mL plastic
beaker, placed on an electronic balance (HF200,
A.N.D., Tokyo, Japan) connected to a personal
computer (Macintosh iBook G3, Apple Computer,
Inc., Cupertino, CA) via a multiport controller(Maclab/400, AD Instruments, Castle Hill, Australia). The cumulative weight of the collected urine
was then monitored. Voided volume and frequency
were sampled by the computer every 150 s for 24 h,and the data were stored on hard disc. Monitoring
per each group started at 1000. During the 24-hobservation period, the rats were given water but no
food.

Materials and Methods
Animals
All animal experiments were performed in accordance with the Tottori University Committee
Guidelines for Animal Experimentation. We divided 20 female Wistar rats aged 16 months into 4groups of 5 animals each. A silastic tube (inside diameter = 2.5 mm, length = 30 mm, Kaneda Medix,
Osaka, Japan, catalog number 100-2N) containing
sesame oil (Sigma-Aldlich, St.Louis, MO, catalog
number S3547) was subcutaneously planted in 3
groups. Ovariectomies were not performed in anygroup. Group I rats received no treatment. Groups
II and III had implantation done with tubes containing 2.50 mg of estradiol (E2) for 4 weeks and 8
weeks, respectively. Group IV rats were implanted
only with sesame oil without E2 for 8 weeks by thesame methods as Groups II and III (Ohata, 2002).
All animals were kept in an air-conditioned room
lighted 12 h a day and allowed access to food andwater ad libitum. At the end of experiment, micturition behavior in a metabolic cage was monitored
and a cystometry under urethane anesthesia was
performed. After that, blood was taken to measureplasma E2.

Cystometry
Cystometry was performed under urethane anesthesia (1.0 g/kg subcutaneously using a 24-G catheter(Terumo, Tokyo, catalog number SR-OT2419C)
inserted into the apex of the bladder dome to recordpressure and ﬁll the bladder with physiological saline. External bladder ﬁlling was carried out using
an infusion pump (5200, TOP, Tokyo) at a constantrate of 0.4 mL/min until voiding was detected. A
cystostomy catheter was connected to an externalpressure transducer (P2310, Gould, Eastlake, OH)
to measure intravesical pressure, which was recorded on a Macintosh Powerbook G3 via a bridge amplifier (ML112, AD Instruments) and a multiportcontroller Maclab/400. During voiding, ﬁlter paper
was carefully placed at the meatus to absorb the
voided urine without spillage, and the voided volume was weighed. The following parameters were
evaluated: bladder capacity, bladder compliance,
maximum detrusor pressure, voided volume and residual urine volume. Maximum detrusor pressure
was deﬁned as instantaneous pressure minus postcontraction resting pressure.

Measurement of plasma E2
Plasma E2 concentrations were assessed by radioimmunoassay with a Tokyo SRL kit. After the rats
were anesthetized with a subcutaneous injection of
1.0 g/kg urethane, a 2.5 mL amount of blood was28

Effect of E2 on bladder function
Table 1. Plasma E2 levels
Group

Plasma E2 level (pg/mL)

I

13.94 ± 3.85

II38.58 ± 6.08

III
IV

*

The number of micturitions per day significantly
decreased in Group III compared with Group IV.
However, there were no differences in volume excreted per day between those groups. The average
voided volume in Groups II and III significantly
increased compared with that in Group IV (Table 2).

***

48.86 ± 9.19*

Voiding behavior study

19.32 ± 8.46

Shown are mean ± SD.
* Signiﬁcant difference: P < 0.05.

Cystometry
Bladder capacity signiﬁcantly increased in the E2replaced groups compared with the other groups,
respectively. Voided volume signiﬁcantly increasedin the E2-replaced groups compared with that inGroup IV. However, bladder compliance showedno signiﬁcant differences in the placebo or untreated groups (Table 3).

Statistical analysis
Statistical comparison of differences between
groups was performed using analysis of varianceand the multiple comparison Fisherʼs test; P < 0.05
was considered signiﬁcant.
Results

Discussion

Plasma E2 levels

Wistar rats have a fertility period that lasts until
approximately 15 months, at which time the estrous
cycle ceases (Handa et al., 1987; Shulman et al.,
1987; Albert et al., 1991). Albert et al. (1991) foundthat a plasma E2 level of 15 pg/mL was slightly
below the mean level of E2 present throughout the
estrous cycle in Wistar rats that maintained normal

Plasma E2 levels in the replaced groups were signiﬁcantly higher than in the untreated and placebogroups, respectively. Between the replaced groups,
Group III showed significantly higher levels inplasma E2 than Group II. There was no signiﬁcantdifference between Groups I and IV (Table 1).
Table 2. Comparison of voiding behavior
Group

Volumeexcreted per day(mL)

Number of
micturitions
per day

Averagevoided volume(mL)

Maximum
voided volume(mL)I

15.83 ± 8.42

17.6 ± 3.1

0.87 ± 0.35

1.93 ± 0.66

II

17.87 ± 6.18

16.4 ± 2.9

1.06 ± 0.22

2.44 ± 0.49

III

14.70 ± 3.50

14.2 ± 2.7

1.01 ± 0.16

IV

14.11 ± 2.56

18.8 ± 3.7*

0.79 ± 0.15**

1.69 ± 0.36
2.26 ± 0.41

Shown are mean ± SD.
* Signiﬁcant difference: P < 0.05.
Average voided volume = (volume excreted per day) divided by (number of micturitions per day)29*

N. Kobayashi et al.

Table 3. Comparison of urodynamic parameters
Group

Bladder
capacity(mL)

Maximum
detrusor pressure(kPa)

Voidedvolume(mL)

Residual
urine volume(mL)

Bladdercompliance(mL/kPa)I

0.38 ± 0.09

25.4 ± 3.4

0.32 ± 0.09

0.13 ± 0.02

0.05 ± 0.02

0.39 ± 0.75

0.09 ± 0.07

0.08 ± 0.02

II
III
IV

*
*

0.52 ± 0.14
0.58 ± 0.09
0.40 ± 0.11**

28.3 ± 2.4*

30.9 ± 4.9

0.36 ± 0.10

30.7 ± 1.5

0.26 ± 0.02**

0.15 ± 0.04
0.22 ± 0.09*

0.07 ± 0.01
0.05 ± 0.03

Shown are mean ± SD.
* Signiﬁcant difference; P < 0.05.

difference remains to unknown, but the parametersof maximum micturition had little reference to thechange of voiding behavior for E2 replacement.
Along with that, cystometry demonstrated that E2replaced groups experienced signiﬁcantly increased
bladder capacity and voided volume compared withGroup IV. E2-replaced groups also showed an increased tendency toward bladder compliance, but
this was not signiﬁcant in our study. Furthermore,
we observed a signiﬁcant difference in maximum
detrusor pressure between Groups I and III, andin residuals urine volume between Groups II and
IV, which might be caused by E2 replacement.
Because there was an age-based bias in sampling
the above groups, intra-group examination of E2
effects was statistically inappropriate. The resultsof voiding behavior and cystometry showed an
improvement in bladder function, correlatively.
There was no signiﬁcant difference in urodynamic
parameters between Groups II and III. The termof E2 replacement affected the plasma E2 levels;
however, the plasma E2 levels after 4 weeks of E2
replacement showed equivalent improvement of
bladder function to those after 8 weeks of replacement.
According to previous reports, ovariectomized
animals had a decrease in the smooth muscle density in the bladders (Hashimoto et al., 1999), and E2replaced groups showed increases in the smoothmuscle content of the bladder (Rocha et al., 2002;

body weight. In the present study, plasma E2 levels
in Group I were slightly lower than 15 pg/mL, as
expected; in Group IV, the levels just exceeded 15
pg/mL, which were slightly higher than expected
at such an age. This may have been caused by the
tube plantation, but the difference between groups
was not signiﬁcant. Groups II and III, which received estrogen replacement, showed significant
increases in plasma E2 levels, achieving levels ofover 30 and 40 pg/mL, respectively. Therefore, theE2 replacement method in this experiment provided
adequate levels for studying the effects of E2 on rat
bladders, as reported (Shulman et al., 1987; Albert
et al., 1991). In consideration of the signiﬁcant difference observed between Groups III and IV, theincrease in plasma E2 levels seemed to be causedby E2 replacement rather than tube plantation.
Voiding behavior changes with age, as demonstrated by increases in voiding frequency (Chunet al., 1988). The present study on voiding behavior proved no differences in volume excreted perday. The number of daily micturitions signiﬁcantly
decreased, and average voided volume increasedin Group III compared with that in Group IV. It
stands to reason that frequent urination improved
due to an increase in average micturition. In thepresent Wistar rats, E2 replacement affected theimprovement of bladder function. The difference inmaximum voided volume was signiﬁcant between
Groups II and III. The underlying cause of this30Effect of E2 on bladder function

Hikita et al., 2004). The increase in smooth muscles of the bladder may improve bladder contraction. These researchers supposed that E2 replacement improved voiding behavior and urodyanamic
parameters.
E2 replacement therapy might prevent apoptosis in the bladder and would influence voiding
behavior (Cockayne et al., 2000; Wotherspoon andWinter, 2000; Ohata et al., 2002). Ohata et al.
(2002) studied effects of E2 on apoptosis, but theyobserved no significant difference in the dose or
replacement term of E2. In the present study, weobserved no difference in the term of E2 replacement. Effects on the dose of E2 need to be studied
further.
The results obtained suggest that hormone
replacement therapy may improve the problem ofurgent and frequent urination in older women.

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Received December 6, 2004; accepted January 7, 2005
Corresponding author: Ikuo Miyagawa , MD

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