The Effect of Ovariectomy and Long-term Estrogen Replacement on Bladder Structure and Function in the Rat

0022-5347/02/1683-1265/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 168, 1265–1268, September 2002 Printed in U.S.A.

DOI: 10.1097/01.ju.0000023406.93873.08

THE EFFECT OF OVARIECTOMY AND LONG-TERM ESTROGEN REPLACEMENT ON BLADDER STRUCTURE AND FUNCTION IN THE RAT NICOLE FLEISCHMANN, GEORGE CHRIST, THERESA SCLAFANI

AND

ARNOLD MELMAN

From the Division of Urologic Research, Albert Einstein College of Medicine, The Bronx, New York

ABSTRACT

Purpose: The use of estrogen replacement therapy for treating postmenopausal urinary incontinence is a controversial topic. We examined the behavioral, cystometric and histological changes that occur with long-term estrogen depletion and supplementation in rat bladders to determine the role of menopause in lower urinary tract dysfunction. Materials and Methods: A total of 40 female Sprague-Dawley rats were placed into 1 of 3 groups, including bilateral ovariectomy, bilateral ovariectomy plus estrogen replacement and control. The estrogen replaced group received a 0.25 mg. 16-week sustained release pellet (Innovative Research of America, Sanasota, Florida) placed subcutaneously. After surgery voiding frequency and volume were measured in 24-hour periods by placing animals in metabolic cages. After 16 weeks the rats underwent catheterization and continuous cystometry. The bladder was then removed and stained with Gomori trichrome. The collagen-to-smooth muscle density ratio was calculated for each specimen using current imaging software. Results: There was no significant difference in voiding patterns in the 3 groups, as measured by volume and voiding frequency. Cystometric data showed a trend toward higher voiding pressure, threshold pressure, baseline pressure and mean inter-voiding pressure in the ovariectomy group compared with the estrogen and control groups, although there was no statistical significance. Histological studies showed a higher mean collagen-to-smooth muscle ratio plus or minus standard deviation in the ovariectomy group (0.807 ⫾ 0.204) than in the ovariectomy plus estrogen replacement (0.709 ⫾ 0.118) and control (0.700 ⫾ 0.129) groups (p ⬍0.05). Furthermore, when histological and cystometric data were compared for individual samples, we found a direct correlation of mean inter-voiding pressure (a measure of bladder instability) with the collagento-smooth muscle ratio (p ⬍0.05). Conclusions: Long-term estrogen replacement is beneficial for treating postmenopausal urinary incontinence. KEY WORDS: bladder; urinary incontinence; estrogen replacement therapy; menopause; rats, Sprague-Dawley

Urinary incontinence is a debilitating problem that is particularly prevalent in elderly women. The changes in bladder function associated with the postmenopausal state, such as detrusor instability, stress urinary incontinence and a propensity toward urinary tract infection, are believed to be due in part to estrogen deficiency.1, 2 The tissues of the lower urinary tract and pelvic floor are known to be estrogen sensitive because estrogen receptors are located throughout the bladder and urethra.3, 4 It is hypothesized that estrogen replacement therapy may be useful for urinary incontinence for several reasons. In women with stress urinary incontinence one may expect estrogen to reverse the effects of urethral atrophy, improving coaptation and, therefore, increasing urethral closure pressure. Furthermore, because estrogen has also been implicated in raising the sensory threshold of the bladder to cholinergic stimulation, estrogen replacement therapy may be equally useful for detrusor instability.5, 6 To date research on the success of estrogen replacement therapy for postmenopausal bladder dysfunction has been laden with controversy. Many studies are based on subjective reports. As early as 1941, Salmon et al reported successful outcomes in postmenopausal women treated with intramuscular estrogen therapy.7 Some investigators reported similar results,8, 9 while others found no improvement in patient perception of the condition.10 There have been few randomAccepted for publication March 28, 2002.

ized, placebo controlled studies measuring objective parameters of bladder dysfunction, for example the number of incontinent episodes, urodynamics or urethrocystometry. Fantl et al studied 83 postmenopausal women with known stress urinary incontinence or detrusor instability who were treated with estrogen therapy or placebo.5 They reported no significant changes in incontinent episodes. In a similar study Jackson et al performed cystometric analysis and urethral profilometry in 67 females before and after 6 months of estrogen replacement.11 They also found no significant differences in the groups. Animal studies have helped to shed some light on the role of estrogen in bladder function. Several in vitro studies of isolated detrusor tissue from ovariectomized rats have shown changes in the contractility of estrogen deprived specimens exposed to various stimulations, for example carbachol, electric field stimulation, 5-hydroxytriptamine and so forth.12–14 Palea et al showed that the bladder and urethra of estradiol pretreated, ovariectomized rats had much lower sensitivity to carbachol stimulation than untreated tissues.15 According to that group this finding explains detrusor instability in estrogen depleted bladders. In contrast, Longhurst et al found that estrogen treatment actually increases detrusor sensitivity and improves bladder contraction.16 A major difference in this study and that of Palea et al5 was the time during which the rats received estrogen therapy (2 to 4 months16 versus 5 days5).

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There have been few in vivo studies done in this area and few to no long-term treatment studies. In the same 1992 study Longhurst et al observed the effects of ovariectomy and estrogen treatment on voiding patterns.16 Rats with 2 to 4 months of therapy were placed in metabolic cages and monitored for volume intake and output as well as frequency of voiding episodes. They found that there was no significant difference in voiding behavior in ovariectomized and untreated rats. To our knowledge there have been no long-term (greater than 4 weeks of treatment) studies to date of urodynamic changes associated with estrogen depletion and replacement therapy. In the current study we measured the behavioral, cystometric and histological changes that occur with longterm estrogen treatment in rat bladders. We believe that this long-term treatment may enable us to assess accurately the role of menopause in lower urinary tract dysfunction. MATERIALS AND METHODS

Experimental groups. A total of 40 Sprague-Dawley female rats weighing 250 to 300 gm. were placed into 1 of 3 groups, including sham operated, bilateral ovariectomy and bilateral ovariectomy plus estrogen replacement groups. The latter 2 groups underwent bilateral ovariectomy under anesthesia using intraperitoneal injection of sodium pentobarbital (35 mg./kg.). At ovariectomy the ovariectomy plus estrogen group received a 0.25 mg. 17␤-estradiol sustained release pellet placed subcutaneously between the shoulder blades. These tablets sustained serum estradiol levels at greater than 100 pg./ml. for a 120-day period. The ovariectomy group received a placebo pellet or no pellet. Animals in all 3 groups underwent voiding studies during the 16-week period until catheterization and subsequent urodynamic testing. The animals were then sacrificed and the bladder was removed for histological analysis. Voiding studies. Voiding behavior was studied beginning 5 weeks after ovariectomy. Each rat was placed in an individual metabolic cage (Nalgene Co., Rochester, New York) suspended over an FT03 displacement transducer (Grass Instruments, Quincy, Massachusetts), which measures liquid with an accuracy of 0.1 ml. During the 12-hour observation period they were given water but no food. Intake and output was recorded as well as the volume and frequency of voiding episodes on a Macintosh computer using MacLab software, V3.4 (Ad Instruments, Grand Junction, Colorado). At the end of the 16-week period the animals underwent cystometric analysis. For in vivo cystometry 16 weeks after ovariectomy the animals underwent bladder catheterization. After anesthetic induction the ventral abdominal wall and perineum were shaved with an electric shaver and cleaned with povidone-iodine. A lower midline incision was made and the bladder was identified. A small incision was made in the bladder dome and a PE-50 Intramedic polyethylene catheter (Becton Dickinson, Franklin Lake, New Jersey) was inserted and secured with a 5-zero silk suture. The catheter was then tunneled subcutaneously and brought out through an orifice made in the animal back. The abdomen was closed with 3-zero silk sutures. To prevent infection the rats received a single injection of sulfadoxine (24 mg./kg.) and trimethoprim (4.8 mg./kg.). At 3 days after surgery animals underwent cystometric anal-

yses since it has been shown to be the optimal period for recovery and investigation.17 These studies were performed in conscious, unanesthetized, freely moving rats. The bladder catheter was connected to a 2-way valve that was in turn connected to a pressure transducer and infusion pump. The pressure transducer was connected via an ETH 400 transducer amplifier (CB Sciences, Dover, New Hampshire) to a MacLab/8e data acquisition board (AD Instruments, Houston, Texas). Realtime display and recording of pressure measurements were performed on a Macintosh computer. The pressure transducers and data acquisition board were calibrated in cm. water before each experiment. Saline solution was infused for 2 hours at a rate of 10 cc per hour. Measurements were made by identifying 3 consecutive voiding cycles of approximately equivalent duration. Bladder function was evaluated by certain urodynamic criteria, including bladder capacity (volume of infused saline at voiding), baseline pressure (lowest bladder pressure during cystometry), threshold pressure, inter-voiding pressure (mean bladder pressure between voids), mean inter-voiding pressure (inter-voiding pressure minus baseline pressure as a measure of bladder pressure variability and detrusor instability), voided volume (urine volume voided during micturition) and voiding frequency (number of voids in 1 hour). At the end of the experiments all animals were sacrificed with an intraperitoneal injection of pentobarbital. For in vitro studies hematoxylin and eosin staining was done on frozen tissue sections to examine tissue structure and determine tissue health. The bladder was removed, the tissue was sectioned on a frozen cryostat at 14 ␮m. and the sections were placed on histology slides. Specimens were cut in cross section from 3 areas of the bladder labeled base, mid and dome or in sagittal sections. The slides were stored at ⫺20C. At staining the sections were first rehydrated with 0.1 M. phosphate buffered saline, pH 7.4, mixed with 0.3% Triton X-100. The standard Mayer hematoxylin and eosin staining procedure was followed. Subsequently Gomori trichrome staining of frozen tissue sections was done to quantify connective tissue-tosmooth muscle ratios. The standard 1-step Gomori trichrome staining procedure, which stains connective tissue green and smooth muscle red, was followed. The sections were then coverslipped with permount and examined with a ⫻2 lens on an epiflourescence microscope (Nikon, New York, New York) using conventional optics. Images were captured with a color spot charge coupled device camera (Diagnostic Instruments, Sterling Heights, Michigan) and a Dell Dimension XPS R350 computer (Dell, Round Rock, Texas) with Image Pro Plus software (Media Cybernetics, Silver Springs, Maryland). Each of the specimens was fully analyzed in 2 or 3 separate areas depending on whether the sample was a sagittal cut or cross section. Using Photoshop 5.0 software (Adobe, San Jose, California) the images were analyzed for red and green pixel areas, as defined by a set range of pixel intensity for red and green. The mean green-to-red ratio was calculated to determine connective tissue-to-smooth muscle density per sample. This method was described by Kim et al in their study of ureteropelvic junction obstruction.18 Statistical analysis. Calculations were done using Sigmastat software (APSS Inc., Chicago, Illinois). Comparisons of groups were made using 1-way analysis of variance and the Fisher least squares difference method. The Pearson product moment correlation test was used for comparing histological and urodynamic parameters with p ⬍0.05 suggesting a correlation of variables.

TABLE 1. Voiding studies 4 months after ovariectomy and estrogen replacement

Sham operation Ovariectomy Ovariectomy ⫹ estrogen

Mean No. Voids/24 Hrs. ⫾ SD

Av. Vol. ⫾ SD (ml.)

4.50 ⫾ 1.73 3.50 ⫾ 3.21 5.00 ⫾ 0.82

2.20 ⫾ 1.87 2.82 ⫾ 1.36 1.620 ⫾ 0.967

RESULTS

Voiding and cystometric studies. Animals in the ovariectomy plus estrogen group showed no significant difference from ovariectomy plus placebo or sham operated animals in terms of voiding frequency or voided volume during a 12-hour

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EFFECT OF OVARIECTOMY AND ESTROGEN REPLACEMENT ON BLADDER TABLE 2. Cystometric analysis 4 months after ovariectomy and estrogen replacement Mean ⫾ SD

Micturition pressure (cm. H2O) Threshold pressure (cm. H2O) Basal pressure (cm. H2O) Inter-voiding pressure Mean inter-voiding pressure (intervoiding pressure-basal pressure) Bladder capacity (ml.) Micturition vol. (ml.) Voiding frequency/2 hrs.

Sham

Bilat. Ovariectomy

Bilat. Ovariectomy Plus Estrogen

62.90 ⫾ 17.22 25.82 ⫾ 10.61 15.30 ⫾ 7.31 22.99 ⫾ 8.99 7.69 ⫾ 2.55

85.40 ⫾ 36.11 43.22 ⫾ 27.29 33.38 ⫾ 30.14 42.27 ⫾ 29.64 8.96 ⫾ 5.18

66.61 ⫾ 19.93 28.15 ⫾ 12.22 16.00 ⫾ 10.21 24.01 ⫾ 11.93 7.71 ⫾ 2.51

2.03 ⫾ 1.73 2.03 ⫾ 1.73 9.5 ⫾ 4.32

1.47 ⫾ .63 1.36 ⫾ .67 13.06 ⫾ 8.82

1.43 ⫾ .59 1.46 ⫾ .68 11.85 ⫾ 8.48

period (tables 1 and 2). For each urodynamic parameter measured there was no difference among the ovariectomy, ovariectomy plus estrogen and sham operated groups (fig. 1). Mean voiding pressure, threshold pressure, baseline pressure, inter-voiding pressure, mean inter-voiding pressure and voiding frequency were notably higher in the ovariectomy group than in the ovariectomy plus estrogen or sham operated groups. However, due to tremendous variation in the ovariectomy group these values did not reach statistical significance. Bladder capacity and voided volume remained unaffected by estrogen replacement or ovariectomy at 4 months. Histological analysis. Gomori trichrome staining of tissue specimens colored connective tissue green and smooth muscle red (fig. 2). The mean green-to-red ratio plus or minus standard deviation was calculated to be significantly higher in the ovariectomy group (0.807 ⫾ 0.204) than in the ovariectomy plus estrogen (0.709 ⫾ 0.118) or sham operated (0.700 ⫾ 0.129) groups (p ⬍0.05). There was no difference in the ovariectomy plus estrogen and sham operated groups. When the results of histological samples were compared with corresponding cystometric data, a direct correlation was found of mean inter-voiding pressure with the green-to-red ratio (p ⬍0.05, fig. 3). No other parameters correlated. DISCUSSION

The current study shows how hormonal manipulation can affect bladder architecture. The increased connective tissueto-smooth muscle tissue ratio observed in the bladders of rats that were ovariectomized without estrogen replacement is evidence of a quantitative structural change related to estrogen deprivation. These findings are consistent with those of Eika et al, who reported an increase in the collagen content of bladder strips of rats ovariectomized for 24 months by measuring hydroxyproline.19 Similarly Hashimoto et al found decreased smooth muscle density in the bladder of ovariectomized animals.20 Susset et al studied bladders at

FIG. 1. OVX, bilateral ovariectomy. ⫹E, plus estrogen replacement. SHAM, sham operation. MP, mean voiding pressure. TP, threshold pressure. BP, baseline pressure. IP, inter-voiding pressure. MIP, mean inter-voiding pressure. BC, bladder capacity. MV, voided volume. Vf, voiding frequency.

FIG. 2. Digital images of specimens. Green areas indicate collagen. Red areas indicate smooth muscle. Gomori trichrome stain, reduced from ⫻2. OVX, bilateral ovariectomy. ⫹E, plus estrogen replacement.

autopsy and found increased collagen content in those of women older than 50 years old compared with men and younger women.21 In the current study bladder instability correlated with increased collagen deposition. Increased collagen deposits throughout the bladder could certainly affect the functional properties of the lower urinary tract by interfering with bladder contraction, decreasing compliance and even changing the sensory threshold for cholinergic stimulation, as in women with detrusor instability. It is likely that this infiltration compromises the ability of the smooth muscle fascicle to perform a normal contraction. Neurohistological studies have shown a decrease in nerve density and increase in collagen deposition in neurogenic bladders which may manifest as interference in the electromechanical transmission of bladder contraction.22 To our knowledge the mechanism by which estrogen may protect the bladder against collagen formation is not yet understood. There is evidence that estrogen replacement exerts its cardioprotective action by preventing the proliferation of fibroblasts and collagen deposition in the blood vessels

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These findings indicate a benefit of estrogen replacement for treating postmenopausal urinary incontinence. REFERENCES

FIG. 3. Scatterplot shows correlation of bladder instability according to mean inter-micturition pressure (MIP) with collagen-tosmooth muscle ratio.

of the heart and this action may be via the endothelial release of nitric oxide.23 Whether there exists a similar mechanism in the bladder is the subject of further investigation. In our study we found that the connective tissue-to-smooth muscle ratio directly correlated with mean inter-voiding pressure, which is a urodynamic parameter measuring bladder instability. However, we were unable to show a difference in voiding frequency or volume in a 12-hour period in animals that received estrogen replacement or were estrogen deprived. Similar results were reported by Longhurst et al.16 Thus, at least on the behavioral level ovariectomized animals do not show signs of urinary incontinence. Cystometric studies also failed to reveal a difference in groups in the parameters used to measure bladder instability. Although there was a trend toward greater detrusor instability as measured by voiding pressure, baseline pressure and mean intervoiding pressure, the large standard deviation in the bilateral ovariectomy group prevented statistical significance. This wide range of values was not seen in the other 2 groups, suggesting that there may be an appropriate interval or threshold of structural damage necessary to produce bladder dysfunction. It is likely that bladder has tremendous reserve and some animals had reached a threshold for bladder decompensation, while others were able to compensate for the changes in bladder structure seen on the microscopic level. Clearly the research to date on the usefulness of estrogen replacement is far from conclusive. There are numerous discrepancies in the data, which complicate the issue of the role of estrogen in bladder structure and function. These differences may be explained by the lack of randomized, placebo controlled studies and the variation in treatment duration, especially on the experimental level. One cannot stress enough the importance of long-term estrogen replacement therapy when performing a relevant study. Changes that occur in the human body during the postmenopausal state are certain to be of a chronic nature and an accurate animal model should consider this principle. CONCLUSIONS

Long-term estrogen deprivation causes significant changes in bladder architecture that can be characterized by an increased collagen-to-smooth muscle ratio. These structural changes are associated with greater bladder instability and are not observed in the bladder of estrogen replaced rats.

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