Yonago Acta medica 2004;47:81–89

Effect of Estrogen Replacement Therapy on BladderCirculation in Old Female Rats
Katsuya Hikita, Naoto Kobayashi and Ikuo Miyagawa
Division of Urology, Department of Surgery, School of Medicine, Tottori University Facultyof Medicine, Yonago 683-8504 JapanThe effect of estradiol (E2) on the urinary bladder of old female rats was investigated byexamining the histology and blood flow in the bladders. A total of 20 female Wister rats
aged 16 months were divided into 4 groups: Group I, 5 normal controls; Group II, 5 rats
treated with E2 for 4 weeks; Group III, rats treated with E2 for 8 weeks; and Group IV,
5 rats receiving a placebo for 8 weeks. After the treatment, we removed the bladders,then weighed and stained them with hematoxylin and eosin. The muscle content wasanalyzed with the Elastica-van Gieson method, and the number of blood vessels with theMassonʼs trichrome method. Blood circulation in the bladders was also measured. TheE2-replaced groups showed higher levels than the other groups in terms of blood flow inthe bladder (20.6 ± 1.8 mL/min for Group II and 23.4 ± 1.5 mL/min for Group III, bothP < 0.05 versus Groups I and IV), muscle content (2.33 ± 0.47 and 3.11 ± 0.48 for Groups
II and III, respectively, both P < 0.05 versus Groups I and IV) and bladder weight (185.3± 6.2 mg and 193.2 ± 23.5 mg for Groups II and III; Group III showed P < 0.05 versus Groups I and IV). Differences in body weight and number of blood vessels amonggroups were not significant. We observed an increase in blood circulation, muscle content and weight of the bladder: E2-replacement therapy positively affected bladder functions.
Key words: bladder; blood flow; estrogen; rat; smooth muscle

Urinary incontinence is one of the most difficult
problems for elderly women to maintain a decent
quality of life. Such changes in bladder function
are produced postmenopausally as detrusor instability, stress urinary incontinence and a propensity
toward urinary tract infection: the dysfunctions areattributed in part to estrogen deficiency (Stenberg etal., 1995; Thom and Brown, 1998). The tissues ofthe lower urinary tract and pelvic floor are known
to be estrogen-sensitive because estrogen receptors
are located throughout the bladder and urethra (Batra and Iosif, 1983; Klutke and Bergman, 1995). It

is hypothesized that estrogen replacement therapy
may be useful for urinary incontinence for several
reasons. In women with stress incontinence, estrogen might reverse the effects of urethral atrophy,improve coaptation and, therefore, increase urethral closure pressure. Because estrogen has also
been linked in increasing the sensory threshold of
the bladder to cholinergic stimulation, estrogen replacement therapy may be equally useful for treating detrusor instability (Ekstrom et al., 1993; Fantlet al., 1996). To date, research on the success ofestrogen replacement therapy for postmenopausal

Abbreviations: E2, estradiol; HE, hematoxylin and eosin; NIH, National Institutes of Health81

K. Hikita et al.
bladder dysfunction has been quite controversial
because many studies were based on subjective
reports. More than 60 years ago, Salmon et al.
(1941) reported successful treatment of postmenopausal women with intramuscular estrogen therapy.
Similar results have been reported elsewhere (Rud,1980).
Animal studies have helped to shed light on
the role of estrogen in bladder function. Longhurstet al. (1992) found that estrogen treatment actually
increases detrusor sensitivity and improves bladder constriction. One study proposed that estrogenic effects may also increase urethral blood flow
(Batra and Iosif, 1983). In that study, the effects
were examined after several days of treatment withrelatively high amounts of estrogen. We decided
to quantitatively assess the effect of long-term estrogen replacement on the smooth muscle, vesicalvessels and blood flow, and muscle content of thebladder in old female rats.

Measurement of plasma E2Plasma E2 concentrations were assessed by radioimmunoassay with a kit from SRL (Tokyo) whenrats were 16 months old in Group I, 17 months oldin Group II and 18 months old in Groups III andIV. After the rats were anesthesized with subcutaneous injection of 1.0 g/kg urethane, an amountof blood (2.5 mL) was taken from the heart andplaced in a cyclone separator to measure the plasma
E2 concentration levels.
Measurement of blood flow in the urinary
bladderWe measured blood flow in the rat bladder with alaser Doppler flowmeter (BRL-100, Bioresearch,Nagoya, Japan). After incision of the abdomen
under anesthesia by subcutaneous injection of 1.0g/kg urethane, we identified the bladder, and emptied and attached a probe to the dome of the bladder (Saito and Miyagawa, 2001) to measure bloodflow for 5 min. Data used were mean ± SE for 1min.

Materials and Methods
All animal experiments were performed in accordance with the Guidelines established by theTottori University Committee for Animal Experimentation. We divided 20 female Wister rats aged
16 months into 4 groups each with 5 animals. Asilastic tube (inside diameter = 2.5 mm, length =30 mm) (Kaneda Medix, Osaka, Japan) containing
sesame oil (Kadoya, Tokyo, Japan) was subcutaneously inserted in the rats of 3 groups (not in thecontrol group). Ovariectomy was not performed
in any group. Group-I rats received no treatment(control group). Group-II rats were administered
2.50 mg of estradiol (E2) dissolved in sesame oil
through a silastic tube for 4 weeks (4-week E2replaced group). Group-III rats were E2-administered for 8 weeks in the same manner as for GroupII (8-week E2-replaced group). Group-IV rats
(placebo group) were treated only with sesame oil
without E2 for 8 weeks by the same methods as forGroups II and III (Ohata, 2002). All animals werekept in an air-conditioned room lighted 12 h a day,and allowed access to food and water ad libitum.

Histological examination of the rat bladder
After the animals were anesthetized with subcutaneous 1.0 g/kg urethane injection, the bladder
was surgically removed from the surrounding tissues and weighed. Each bladder was transected
at the urethra level, sectioned into sagital slices,immediately fixed with 10% formalin, and thenembedded in paraffin. From these paraffin blocks,
10-µm-thick tissue sections were prepared in vertical cross slices. The tissue sections were deparaffinized with xylene, and rehydrated with a graded
series of ethanol. All specimens were stained either with hematoxylin and eosin (HE), Elastica-van
Gieson or Massonʼs trichrome. The Elastica-van
Gieson method distinguishes the smooth muscle
area density from the connective tissue area density, staining smooth muscle yellow and collagen red.
Each section was viewed under a light microscopeat a magnification of 400 times. To analyze the ef-82

Effect of E2 on bladder circulation

fect of estrogen replacement on the smooth muscle
content, color-assisted quantitative image analysis
was used with the Elastica-van Gieson method.
The Massonʼs trichrome method was applied toobserve each section of blood vessels in the smoothmuscle of the bladder linings. Data were obtained
from the 5 animals in each group and averaged.

Statistical analysis
Statistical significances of differences amonggroups were compared with analysis of variance(ANOVA) and Fisherʼs multiple comparison test.
Probability levels less than 0.05 were regarded assignificant.

Color-assisted quantitative image analysis
Results

The stained tissue sections were viewed under amicroscope (BX50, Olympus, Tokyo). The imageof the histologic sections was displayed on a color
monitor (PVM-20M4V, Sony, Tokyo) and printed
with a digital color printer (CP700DSA, Mitsubishi, Tokyo). It was then digitized with a scanner(GT-8000, Epson, Tokyo) operated by a Macintosh
Powerbook G3 (Apple Computer, Inc., Cupertino,CA), and automated image analysis was performed
with National Institutes of Health (NIH) Image 1.55
(Research Service Branch, NIH, Bethesda, MD).
The NIH Image 1.55 system discriminates color
differences in stained tissue sections: We applied
the system to discriminate differentially stained
components of smooth muscle and connective tissues, which were calculated per full screen. At
least 10 different fields were examined from eachtissue section. The ratio of smooth muscle area/connective tissue area was determined for eachanimal.

Plasma E2 levels
Plasma E2 levels ranged from 7.5 to 28.3 pg/mL
in Group I, 35.0 to 57.5 pg/mL in Group II, 35.0 to
57.4 pg/mL in Group III and 9.9 to 19.7 pg/mL in
Group IV. Differences in plasma E2 levels weresignificant between all pairs of Groups, except between Groups I and IV (Table 1).
Bladder and body weight
Bladder weight was significantly higher in GroupIII than in Groups I and IV. Group II showeda higher tendency of mean bladder weight thanGroups I and IV, but there was no statistical significance (Table 1). Differences in body weight
were not significant among all groups, while E2supplied groups showed a lower tendency of mean
body weight.

Table 1. Plasma E2 levels, Bladder weight and Body weightGroupPlasma E2 levels (pg/mL)

Bladder weight (mg)Body weight (g)I

19.3 ± 8.4

164.5 ± 9.1

355.5 ± 32.0

II

38.5 ± 6.1

III
IV

*

*

48.8 ± 9.1**

185.3 ± 6.2*

193.2 ± 23.5

13.9 ± 3.8

164.3 ± 14.0

Each value is mean ± SE.
* Significant difference; P < 0.05.

83

*
*

338.6 ± 27.0
344.8 ± 34.2
349.7± 25.6

K. Hikita et al.

AFig. 1. Sections of rat bladders
stained with the Massonʼs trichrome method (original magnification, × 400).
A: A control rat (Group I).
B: A rat treated with E2
for 4 weeks (Group II).
C: A rat treated with E2
for 8 weeks (Group III).
D: A placebo rat (Group IV).

Figs. 1A and B on p. 84
andFigs. 1C and D on p. 85

B

Table 2. Blood flow and average number of bladder vessels

Blood flow and histological examination ofthe rat bladderGroup

Blood flow (mL/min)Number of vessels

I16.6 ± 1.8

3.6 ± 1.1

II20.6 ± 1.8

Blood flow levels of the bladder in Groups II and
III were significantly (P < 0.05) higher than inGroups I and IV. A longer period of E2 supply
showed an increasing tendency toward blood flow:
blood flow level was higher in the 8-week-suppliedGroup III than the 4-week-supplied Group II (Table2). The difference in number of blood vessels ofthe bladder was not significant, but Groups II andIII tended to show a higher number than Groups Iand IV (Table 2).

III
IV

*

*

23.4 ± 1.5**

18.2 ± 0.4*

4.8 ± 1.3
5.0 ± 1.2
3.6 ± 1.3

Each value is mean ± SE.
* Significant difference; P < 0.05.

84