The 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 and Groups 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). This suggests that E2-replacement therapy positively affects bladder function

Hikita, Katsuya

Kobayashi, Naoto

Miyagawa, Ikuo

Yonago Acta Medica

Tottori University Research Result Repository

Yonago Acta medica 2005;48:27–3127Effect 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 Faculty of 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: un-treated; 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 urody-namic parameters were analyzed.  Particularly, bladder capacity as well as voided vol-ume 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 and Groups 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 void-ed 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), respec-tively.  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). This suggests that E2-replacement therapy positively affects bladder function.  Key words:  bladder function; estrogen; ratUrinary incontinence is a debilitating problem par-ticularly prevalent in elderly women.  Changes in bladder function, associated with such postmeno-pausal states as detrusor instability, stress urinary incontinence, and a propensity toward urinary tract infection, are believed in part to be due to estrogen deﬁciency (Stenberg et al., 1995; Thom and Brown, 1998).  Lower urinary tract and pelvic floor tis-sues are known to be estrogen sensitive because estrogen receptors are located throughout the blad-der and urethra (Batra and Iosif, 1983; Klutke and Bergman, 1995).  For several reasons, it is hypoth-esized that estrogen replacement therapy may be useful in treating urinary incontinence.  In women suffering from stress incontinence, estrogen is ex-pected to reverse the effects of urethral atrophy, improving coaptation, and therefore increasing ure-thral closure pressure (Fantl et al., 1996).  Further-more, because estrogen has also been implicated in raising the sensory threshold of the bladder to cholinergic stimulation, estrogen replacement ther-apy 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 bladder dysfunction has been saddled with controversy be-cause many studies are based on subjective reports. As early as 1941, Salmon et al. (1941) reported suc-cessful outcomes in postmenopausal women treated with intramuscular estrogen replacement therapy. Abbreviation:  E2, estradiol.N. Kobayashi et al.28Other investigators reported similar results (Rud, 1980). Animal studies have shed light on the role of estrogen in bladder function.  Longhurst et al. (1992) found that estrogen treatment actually increases detrusor sensitivity and improves bladder construc-tion.  We studied the urodynamic changes associat-ed with estrogen depletion and replacement therapy. In the current study we measured voiding behavior and cystometry and assessed estrogen treatment.  Materials and MethodsAnimalsAll animal experiments were performed in ac-cordance with the Tottori University Committee Guidelines for Animal Experimentation.  We di-vided 20 female Wistar rats aged 16 months into 4 groups of 5 animals each.  A silastic tube (inside di-ameter = 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 any group.  Group I rats received no treatment.  Groups II and III had implantation done with tubes con-taining 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 the same 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 and water ad libitum.  At the end of experiment, mictu-rition behavior in a metabolic cage was monitored and a cystometry under urethane anesthesia was performed.  After that, blood was taken to measure plasma E2.Measurement of plasma E2Plasma E2 concentrations were assessed by radio-immunoassay 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 was taken from the heart and was placed in a cyclone separator to measure plasma E2 concentration levels. Voiding behavior studyEach rat was placed in a metabolic cage containing a urine collection funnel to record their voiding be-havior.  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, Austra-lia).  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-h observation period, the rats were given water but no food. CystometryCystometry was performed under urethane anesthe-sia (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 record pressure and ﬁll the bladder with physiological sa-line.  External bladder ﬁlling was carried out using an infusion pump (5200, TOP, Tokyo) at a constant rate of 0.4 mL/min until voiding was detected.  A cystostomy catheter was connected to an external pressure transducer (P2310, Gould, Eastlake, OH) to measure intravesical pressure, which was record-ed on a Macintosh Powerbook G3 via a bridge am-plifier (ML112, AD Instruments) and a multiport controller Maclab/400.  During voiding, ﬁlter paper was carefully placed at the meatus to absorb the voided urine without spillage, and the voided vol-ume was weighed.  The following parameters were evaluated:  bladder capacity, bladder compliance, maximum detrusor pressure, voided volume and re-sidual urine volume.  Maximum detrusor pressure was deﬁned as instantaneous pressure minus post-contraction resting pressure.Effect of E2 on bladder function 29Statistical analysisStatistical comparison of differences between groups was performed using analysis of variance and the multiple comparison Fisher sʼ test; P < 0.05 was considered signiﬁcant.  ResultsPlasma E2 levelsPlasma E2 levels in the replaced groups were sig-niﬁcantly higher than in the untreated and placebo groups, respectively.  Between the replaced groups, Group III showed significantly higher levels in plasma E2 than Group II. There was no signiﬁcant difference between Groups I and IV (Table 1).Table 1.  Plasma E2 levels  Group Plasma E2 level (pg/mL) I 13.94 ± 3.85       II 38.58 ± 6.08 III  48.86 ± 9.19 IV  19.32 ± 8.46 Shown are mean ± SD.* Signiﬁcant difference: P < 0.05.Table 2.  Comparison of voiding behavior  Group Volume Number of  Average   Maximum   excreted per day  micturitions voided volume  voided volume  (mL) per day (mL) (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   1.69 ± 0.36 IV 14.11 ± 2.56   18.8 ± 3.7   0.79 ± 0.15   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) Voiding behavior studyThe number of micturitions per day significantly decreased in Group III compared with Group IV. However, there were no differences in volume ex-creted per day between those groups.  The average voided volume in Groups II and III significantly increased compared with that in Group IV (Table 2). CystometryBladder capacity signiﬁcantly increased in the E2-replaced groups compared with the other groups, respectively.  Voided volume signiﬁcantly increased in the E2-replaced groups compared with that in Group IV.  However, bladder compliance showed no signiﬁcant differences in the placebo or untreat-ed groups (Table 3).   DiscussionWistar 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) found that 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 * ********N. Kobayashi et al.30body 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 re-ceived estrogen replacement, showed significant increases in plasma E2 levels, achieving levels of over 30 and 40 pg/mL, respectively.  Therefore, the E2 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 dif-ference observed between Groups III and IV, the increase in plasma E2 levels seemed to be caused by E2 replacement rather than tube plantation. Voiding behavior changes with age, as dem-onstrated by increases in voiding frequency (Chun et al., 1988).  The present study on voiding behav-ior proved no differences in volume excreted per day.  The number of daily micturitions signiﬁcantly decreased, and average voided volume increased in Group III compared with that in Group IV.  It stands to reason that frequent urination improved due to an increase in average micturition.  In the present Wistar rats, E2 replacement affected the improvement of bladder function.  The difference in maximum voided volume was signiﬁcant between Groups II and III.  The underlying cause of this difference remains to unknown, but the parameters of maximum micturition had little reference to the change of voiding behavior for E2 replacement. Along with that, cystometry demonstrated that E2-replaced groups experienced signiﬁcantly increased bladder capacity and voided volume compared with Group IV.  E2-replaced groups also showed an in-creased 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, and in 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 results of 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 term of 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 replace-ment.  According to previous reports, ovariectomized animals had a decrease in the smooth muscle densi-ty in the bladders (Hashimoto et al., 1999), and E2-replaced groups showed increases in the smooth muscle content of the bladder (Rocha et al., 2002; Table 3.  Comparison of urodynamic parameters  Group Bladder Maximum Voided Residual Bladder  capacity  detrusor pressure volume urine volume compliance  (mL) (kPa) (mL) (mL) (mL/kPa)  I   0.38 ± 0.09 25.4 ± 3.4 0.32 ± 0.09  0.13 ± 0.02 0.05 ± 0.02 II  0.52 ± 0.14 28.3 ± 2.4 0.39 ± 0.75  0.09 ± 0.07  0.08 ± 0.02 III  0.58 ± 0.09 30.9 ± 4.9  0.36 ± 0.10  0.15 ± 0.04 0.07 ± 0.01 IV  0.40 ± 0.11 30.7 ± 1.5 0.26 ± 0.02  0.22 ± 0.09 0.05 ± 0.03 Shown are mean ± SD.  * Signiﬁcant difference; P < 0.05.********Effect of E2 on bladder function 31Hikita et al., 2004).  The increase in smooth mus-cles of the bladder may improve bladder contrac-tion.  These researchers supposed that E2 replace-ment improved voiding behavior and urodyanamic parameters. E2 replacement therapy might prevent apop-tosis in the bladder and would influence voiding behavior (Cockayne et al., 2000; Wotherspoon and Winter, 2000; Ohata et al., 2002).  Ohata et al. (2002) studied effects of E2 on apoptosis, but they observed no significant difference in the dose or replacement term of E2.  In the present study, we observed no difference in the term of E2 replace-ment.  Effects on the dose of E2 need to be studied further. The results obtained suggest that hormone replacement therapy may improve the problem of urgent and frequent urination in older women. References  1 Albert DJ, Jonik RH, Gorzalka BB, Newlove T, Webb B, Walsh ML.  Serum estradiol concentra-tion required to maintain body weight, attractivity, proceptivity, and receptivity in the ovariectomized female rat.  Physiol Behav 1991;49:225–231. 2 Batra S, Iosif C.  Female urethra:  a target for estro-gen action.  J Urol 1983:129:418–420. 3 Cockayne AD, Hamilton GS, Zhu MQ, Dunn MP, Zhong Y, Novakovic S.  Urinary bladder hypore-flexia and reduced pain-related behavior in P2X3-deﬁcient mice.  Nature 2000;407:1011–1015. 4 Chun AL, Wallace LW, Gerald MC, Levine RM, Wein AJ.  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The use of estro-gen in the treatment of dysuria and incontinence in postmenopausal women.  Am J Obstet Gynecol 1941;42:845–851.16 Shulman DI, Sweetland M, Duckett G, Root AW. Effect of estrogen on the growth hormone (GH) secretory response to GH-releasing factor in the castrated adult female rat in vivo.  Endocrinology 1987;120:1047–1051.17 Stenberg A, Heimer G, Ulmsten U.  The prevalence of urogenital symptoms in postmenopausal women. Maritatas 1995;22:S17–20.18 Thom DH, Brown JS.  Reproductive and hormonal risk factors for urinary incontinence in later life:  a review of clinical and epidemiologic literature.  J Am Geriatr Soc 1998;46:1411–1417.19 Wotherspoon G, Winter J.  Bladykinin B1 receptor is constitutively expressed in the rat sensory nervous system.  Neurosci Lett 2000;294:175–178.Received December 6, 2004; accepted January 7, 2005Corresponding author: Ikuo Miyagawa , MD

Effect of Long-Term Estrogen Replacement on Bladder Function in Old Female Rats

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Effect of Long-Term Estrogen Replacement on Bladder Function in Old Female Rats

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