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Effect of Ovariectomy on External Urethral Sphincter Activity in Anesthetized Female Rats
Effect of Ovariectomy on External Urethral Sphincter Activity in Anesthetized Female Rats Chen-Li Cheng* and William C. de Groat† From the Division of Urology, Department of Surgery, Taichung Veterans General Hospital (CLC), Taichung, Taiwan, Republic of China, and Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine (WCdG), Pittsburgh, Pennsylvania
Abbreviations and Acronyms 5-HT1A ⫽ 5-hydroxytryptamine1A AP ⫽ active period BD ⫽ bursting duration CA ⫽ contraction amplitude CD ⫽ contraction duration CMG ⫽ cystometrogram E2 ⫽ estradiol EMG ⫽ electromyogram EUS ⫽ external urethral sphincter LUT ⫽ lower urinary tract OVX ⫽ ovariectomy PE ⫽ polyethylene RV ⫽ post-void residual volume SP ⫽ silent period TSP ⫽ total SP VE ⫽ voiding efficiency VT ⫽ volume threshold Submitted for publication September 22, 2010. Study received Taichung Veterans General Hospital institutional animal care and use committee approval. Supported by Grant NSC97-2314-B-075A-009MY3 from the National Science Foundation, Taiwan, Republic of China. * Correspondence: Division of Urology, Department of Surgery, Taichung Veterans General Hospital, 160, Section 3, Taichung-Kang Rd., Taichung, Taiwan, Republic of China (telephone: 886-42374-1215; FAX: 886-42359-3160; e-mail; [email protected]). † Financial interest and/or other relationship with Endo Pharmaceutical, Pfizer, Medtronic, Ethicon, Astellas, Eli Lilly and Takeda.
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Purpose: The postmenopausal hypoestrogen condition is associated with various lower urinary tract dysfunctions, including frequency, urgency, stress urinary incontinence and recurrent urinary infection. We determined whether hypoestrogen induced lower urinary tract dysfunction after ovariectomy is also associated with an alteration in external urethral sphincter activity. Materials and Methods: Bilateral ovariectomy was performed in female SpragueDawley® rats and sham operated rats served as controls. Transvesical cystometry and external urethral sphincter electromyogram activity were monitored 4, 6 and 12 weeks after sham operation or bilateral ovariectomy and at 6 weeks in bilaterally ovariectomized rats treated with estrogen. Results: The micturition reflex was elicited in sham operated and bilaterally ovariectomized, urethane anesthetized animals. Post-void residual urine increased and voiding efficiency decreased in rats with 4 to 12 weeks of bilateral ovariectomy. The silent period of external urethral sphincter electromyogram activity was shortened significantly and progressively at increased times after bilateral ovariectomy. These effects were prevented by estradiol treatment. Conclusions: As evidenced by shortening of the external urethral sphincter electromyogram silent period in ovariectomized rats, the disruption of coordination between the external urethral sphincter and the detrusor muscle could decrease urine outflow and in turn voiding efficiency. Estrogen replacement reverses these changes, suggesting that the central pathways responsible for detrusor-sphincter coordination are modulated by gonadal hormones. Key Words: urethra, ovariectomy, estrogens, menopause, urination disorders THE LUT and genital organs, which share a common embryological origin, express estrogen and progesterone receptors, and show physiological responses to these hormones.1–3 Postmenopausal hypoestrogen status elicits anatomical and physiological changes in LUT4 and may be associated with various LUT dysfunctions, including frequency, urgency, urge incontinence, stress urinary incontinence, detrusor overactivity and recurrent urinary tract infections.5 The influence of gonadal hormones on
LUT function is likely mediated via multiple effects on neural control, vascular supply, and detrusor muscle cell size and number as well as by connective tissue density and distribution.4 –7 Most groups have focused on functional and morphological changes in the bladder and urethral smooth muscle in the hypoestrogen condition after menopause or OVX in human and animal models, respectively.2,4,6,7 We explored the effect of OVX on EUS activity and its coordination with the
0022-5347/11/1861-0334/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 334-340, July 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.03.035
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EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
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Bilateral OVX was performed via a midline abdominal approach using isoflurane anesthesia and aseptic techniques. In sham operated controls the 2 ovaries were visualized but not removed. The animals were treated with antibiotic (ampicillin 150 mg/kg intramuscularly daily) for 2 to 3 days to prevent infection after surgery.
where EUS muscle fibers were identified. The recorded EUS EMG was attributable to striated muscle since the activity was eliminated after neuromuscular blockade with pancuronium bromide (Organon, Oss, The Netherlands).9 A PE 60 tube with a 1.0 mm inner diameter and 1.5 mm outer diameter was inserted in the bladder lumen. The bladder end of the PE tube was heated to create a collar and passed through a small incision at the apex of the bladder dome. A suture was tightened around the collar of the tube. The abdominal wall was then closed with nylon suture. The PE tube was in turn connected via a 3-way stopcock to an infusion pump and a pressure transducer. The system was then filled with physiological saline. Urodynamic examination usually began 3 to 4 hours after anesthesia induction. After the bladder was emptied transvesical cystometry was done at an infusion rate of 0.123 ml per minute with physiological saline at room temperature. The urethra was open and fluid could be evacuated during micturition. The infusion pump was turned off after induction of a voiding contraction. RV was measured by empting the bladder by pressure on the abdominal wall. Various parameters were measured, including VT (saline volume sufficient to induce bladder contraction exceeding 15 cm H2O), CA (maximal intravesical pressure during voiding), CD, RV (saline volume withdrawn through the intravesical catheter after voiding) and VE, expressed as a percent using the formula, VE ⫽ voided volume (VT ⫺ RV)/VT ⫻ 100. Investigators performing EUS EMG activity analysis were blinded to rat status. As previously described,13 various EUS EMG parameters were measured, including average BD, SP and AP (fig. 1), TSP during each void and the ratio of BD to CD, expressed as a percent. Bursting was measured during the time when EMG was completely quiescent between bursts (fig. 1), which occurred when intravesical pressure began to decrease during voiding. Before this time some phasic EMG activity was detected but an SP was not obvious (fig. 1). At least 5 transvesical CMGs were done per animal. All parameters were calculated with AcqKnowledge™. Calculated data were compiled in spreadsheets using Excel®. EUS EMG activity was displayed on a storage oscilloscope and a paper recorder along with bladder pressure.
E2 Treatment
Statistical Analysis
The rats were randomly assigned to hormone replacement with 17-E2 (0.05 mg as a 21-day time release E2 pellet) (Innovative Research of America, Sarasota, Florida). The pellet was implanted subcutaneously at the mid scapular level 3 weeks after OVX.
Results are shown as the mean ⫾ SE. Data were tested for normal distribution using the Shapiro-Wilk test and found to be not normally distributed (p ⬍0.01). Thus, associations between sham operated and OVX rats were assessed by the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test with the p value adjusted for the 4 comparisons using the Bonferroni correction. Differences were considered statistically significant at p ⬍0.05.
detrusor muscle during voiding in anesthetized female rats. In previous experiments at various laboratories groups reported that EUS EMG in rats shows tonic activity during bladder filling and bursting activity during voiding.8 –12 Bursting can be divided into APs and SPs, and analyzed using various parameters, including the duration and interval of these periods and of total BD.13 The pattern of EUS EMG activity can be altered by drugs8,10,12,14 and pathological conditions such as spinal cord or pudendal nerve injury while changes in EUS EMG activity correlate with changes in voiding function.8,10,13,15 The current experiments revealed that OVX also produces prominent changes in EUS EMG activity that are linked to decreased VE.
MATERIALS AND METHODS Experimental Protocols Adult female Sprague-Dawley rats were housed individually in a room controlled for temperature, humidity and lighting with lights on from 7:00 a.m. to 7:00 p.m. Rats had free access to food and water. A total of 76 female Sprague-Dawley rats weighing 230 to 320 gm were used in this study. The animals were divided into 7 groups. Groups 1 to 3 underwent sham operation and were studied 4 (11 rats), 6 (12) and 12 (11) weeks postoperatively, respectively. Groups 4 to 6 underwent OVX and were studied at 4 (11 rats), 6 (11) and 12 (10) weeks postoperatively, respectively. Group 7 (10 rats) underwent bilateral OVX, and was treated with 17-E2 replacement for 3 weeks starting 3 weeks after OVX and studied 6 weeks after OVX. The Taichung Veterans General Hospital institutional animal care and use committee approved the study protocol.
OVX Surgery
Physiological Investigation Experiments were done using urethane anesthesia (1.2 gm/kg subcutaneously). The femoral vein was catheterized for fluid administration. Body temperature was maintained between 36C and 38C with a heating lamp. The bladder was exposed via a midline abdominal incision. The rostral half of the pubic symphysis was removed to expose the mid urethra and the EUS. Two fine insulated silver wire electrodes 0.05 mm in diameter with exposed tips were inserted into the lateral sides of the mid urethra,
RESULTS Uterus and Body Weight The mean body weight of rats with 4 to 12-week OVX was significantly larger than that of time matched, sham operated rats 8% to 21%, (p ⬍0.05,
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Figure 1. Bladder (A, C, E and G) and EUS EMG (B, D, F and H) activity recorded during continuous transvesical infusion CMG in anesthetized rat. Reflex bladder contraction, indicated by large, abrupt bladder pressure increase, was accompanied by large amplitude EUS EMG activity (A and B). Vertical calibration represents intravesical pressure in cm H2O (A). Arrow indicates start of saline infusion (A). Asterisk indicates C and D. Reflex bladder contraction recording at more rapid time scale (C and D). Bracket (C) indicates E and F. Tonic EUS EMG activity preceded large intravesical pressure increase and shifted to bursting pattern at bladder contraction peak before voiding onset (E and F). Bracket (E) indicates G and H. Recordings at more rapid time scale show individual EUS EMG bursts composed of APs and SPs (brackets), and small intravesical pressure fluctuations accompanying each burst (G and H).
table 1). The uterus and fallopian tubes appeared atrophied in rats with OVX. The uterine weight of rats with 4 to 12-week OVX was significantly lower than of sham operated rats (18% to 26% of control values, table 1). OVX Effects Bladder activity during transvesical CMG. With the urethral outlet open to allow intravesical fluid to be evacuated during voiding, various parameters of LUT activity and EUS EMG were assessed during slow saline infusion, transvesical CMG. The micturition reflex was elicited in all controls and rats with OVX at different postoperative intervals (4 to 12 weeks). However, some bladder activity parameters changed at different times after OVX. VT, CA and CD did not change 4 to 12 weeks after OVX but RV increased significantly from 0.27 to 0.38 ml starting 4 weeks after OVX (table 2). VE decreased progressively from 57.7% to 43.9% after OVX despite unchanged maximal voiding pressure (table 2).
EUS EMG activity. During continuous infusion CMG before the onset of voiding 2 types of EUS EMG activity were detected. Most rats showed consistent, low amplitude, tonic EUS EMG activity during the filling phase. In some animals this tonic activity increased gradually as infusion volume approached the micturition VT. During bladder contraction the EUS EMG activity markedly increased, consisting of an initial period of tonic activity interspersed with phasic activity in some rats, followed by a bursting pattern of activity characterized by clusters of high frequency spikes (AP) separated by quiescent periods (SP). SP decreased significantly and progressively 21% to 35% at increased times after OVX (table 2 and fig. 2). TSP was significantly increased 4 and 6 weeks after OVX but not at 12 weeks (table 2). There was no difference in AP duration between rats with OVX and controls at 4 weeks but AP duration significantly increased 6 and 12 weeks after OVX (table 2 and fig. 2). BD and the ratio of BD to CD were decreased 13% to 16% 4 and 6 weeks after OVX, respectively, but these changes were not statistically significant (table 2). Effect of Post-OVX E2 Replacement on Bladder Activity Transvesical CMG done after 3 weeks of E2 treatment (0.05 mg as a 21-day time release E2 pellet), which started 3 weeks after OVX, revealed that the VT increased (85%) in E2 treated animals compared to that in rats with 6-week OVX, although OVX did not decrease bladder VT (table 2). The change in VE that occurred after OVX was completely reversed by E2 treatment (table 2). Also, SP, BD and TSP were significantly increased by E2 treatment (table 2 and fig. 2).
Table 1. Effect of OVX and E2 replacement on body and uterine weight Mean ⫾ SE Wt
4 Wks: Control OVX p Value 6 Wks: Control OVX p Value OVX ⫹ 3-wk E2 p Value 12 Wks: Control OVX p Value
No. Rats
Body (gm)
Uterus (mg)
8 9
298.5 ⫾ 16.9 358.1 ⫾ 35.2 0.000*
476.0 ⫾ 98.6 124.3 ⫾ 16.0 0.016*
9 9
335.8 ⫾ 23.8 363.3 ⫾ 27.4 0.002* 326.0 ⫾ 12.2 0.000*
542.0 ⫾ 82.9 140.9 ⫾ 28.1 0.000* 508.5 ⫾ 152.0 0.000*
328.6 ⫾ 22.0 400.7 ⫾ 34.5 0.000*
561.9 ⫾ 93.2 102.5 ⫾ 12.4 0.000*
8
9 8
* Mann-Whitney U test with Bonferroni correction p ⬍0.05.
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0.120 ⫾ 0.010 0.067 ⫾ 0.008 5.63 ⫾ 1.80 3.62 ⫾ 1.18 25.17 ⫾ 7.28
0.093 ⫾ 0.074 ⫾ 4.69 ⫾ 2.65 ⫾ 21.18 ⫾
0.017 0.007 2.22 1.49 7.94
0.002* 0.034* 0.171 0.040* 0.365
0.105 ⫾ 0.028 0.068 ⫾ 0.007 7.03 ⫾ 4.83 4.42 ⫾ 3.75 27.34 ⫾ 10.14
0.349 0.085 0.043* 0.020* 0.051
0.111 ⫾ 0.013 0.068 ⫾ 0.004 5.75 ⫾ 2.96 3.55 ⫾ 1.79 23.52 ⫾ 7.70
0.084 ⫾ 0.079 ⫾ 6.22 ⫾ 3.22 ⫾ 23.17 ⫾
0.012 0.007 4.16 2.26 9.80
0.000* 0.004* 0.705 0.282 0.426
DISCUSSION
* Mann-Whitney U test with Bonferroni correction p ⬍0.05.
0.021* 0.332 0.151 0.047* 0.171 0.022 0.010 2.13 1.17 6.88 0.096 ⫾ 0.072 ⫾ 5.39 ⫾ 3.15 ⫾ 22.98 ⫾ 0.127 ⫾ 0.017 0.066 ⫾ 0.004 6.19 ⫾ 1.26 4.12 ⫾ 0.88 28.09 ⫾ 7.42
0.809 0.654 0.863 0.008* 0.003* ⫾ 0.27 ⫾ 5.76 ⫾ 0.16 ⫾ 0.22 ⫾ 16.92 0.67 36.10 0.43 0.38 43.85 0.49 33.18 0.38 0.08 83.23
No. rats Cystometry: VT (ml) CA (cm H2O) CD (mins) RV (ml) % VE EUS EMG: SP (secs) AP (secs) BD (secs) TSP (secs) % BD/CD
11
⫾ 0.22 ⫾ 5.54 ⫾ 0.05 ⫾ 0.06 ⫾ 8.34
11
0.60 38.68 0.40 0.27 57.74
⫾ 0.22 ⫾ 2.59 ⫾ 0.11 ⫾ 0.19 ⫾ 21.81
0.243 0.016* 0.847 0.007* 0.004*
0.50 32.76 0.38 0.12 75.15
12
⫾ 0.12 ⫾ 4.48 ⫾ 0.09 ⫾ 0.04 ⫾ 8.34
11
0.54 39.09 0.36 0.29 48.64
⫾ 0.20 ⫾ 3.95 ⫾ 0.06 ⫾ 0.15 ⫾ 11.69
0.438 0.016* 0.438 0.002* 0.000*
1.00 31.61 0.41 0.26 74.19
10
⫾ 0.34 ⫾ 6.03 ⫾ 0.10 ⫾ 0.12 ⫾ 10.38
0.004* 0.015* 0.349 0.809 0.001*
0.63 34.60 0.40 0.20 67.91
11
⫾ 0.13 ⫾ 6.13 ⫾ 0.09 ⫾ 0.05 ⫾ 4.62
10
Mean ⫾ SE OVX p Value Mean ⫾ SE OVX Mean ⫾ SE Control p Value Mean ⫾ SE OVX Mean ⫾ SE Control
4 Wks
Table 2. Effect of OVX and E2 replacement on cystometric parameters and EUS EMG activity
6 Wks
Mean ⫾ SE OVX ⫹ 3-Wk E2
p Value
Mean ⫾ SE Control
12 Wks
p Value
EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
The current experiments reveal that rats with OVX showed several voiding abnormalities, including 1) increased RV, 2) decreased VE and 3) an alteration in EUS EMG bursting activity, characterized primarily by a decreased SP duration during micturition. These observations indicate that coordination between bladder and EUS is disrupted by OVX, leading to a decrease in the periods of EUS relaxation, which in turn decreases the bladder emptying efficiency. These effects were reversed by E2 treatment. Previous studies in rats indicated that OVX increased voiding frequency,7,16 suggesting that a decrease in gonadal hormone levels increases the activity of bladder smooth muscle or the neural pathways controlling the bladder. One study also showed that OVX induced an age and time dependent change in urodynamic parameters, including an increase in urethral opening pressure and maximal voiding pressure as well as a decrease in maximal flow rate and voided volume.17 These changes were reversed by treatment with exogenous estrogen for 4 weeks. Our experiments in rats with OVX did not reveal a change in the VT to initiate micturition or in peak intravesical pressure during micturition. However, they revealed decreased VE associated with a decrease in the duration of EUS SPs and TSPs. The change in SP occurred at the earliest time tested after OVX (4 weeks) and increased in magnitude at longer times (6 to 12 weeks). EUS bursting activity reflects the rhythmic opening and closing of the outlet to produce a pulsatile flow of urine, which is common in rodents. Since bursting represents the period of flow through the urethra18 and SP represents the period of maximal urethral relaxation, it is reasonable to conclude that changes in EUS neural control contribute to the inefficient voiding and urinary retention seen in the current experiments. They also likely account for the decrease in the maximal urine flow rate reported by Diep and Constantinou in rats with OVX.17 On the other hand, it is uncertain whether the changes in bladder function after OVX that were reported by others contribute to decreased VE.6,7 OVX significantly increased body weight, an effect that may have increased urine production and produced the small but not significant increase in bladder VT. However, it is unlikely that the change in body weight contributed to the putative neurally mediated changes in EUS EMG activity or VE. SP shortening and decreased BD/CD were detected in chronic spinal cord transected rats, which show detrusor-sphincter dyssynergia, decreased VE and increased RV.13 In spinal cord transected rats the change in EUS EMG activity is clearly due to a central nervous system abnormality. By analogy it seems reasonable to propose that the changes in EUS EMG
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EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
A cmH2O 50
A2
A1 SHAM
0
5 min
B cmH2O 50
B1
A3
B2
5 sec
1 sec
OVX 4W
0
5 min
C cmH2O 50
B3
5 sec
1 sec
C1 OVX 6W
C2
0
5 min
D cmH2O 50
D1
C3
D2
5 sec
1 sec
OVX 12W
0
D3
5 sec
5 min
E cmH2O 50
E1
E2
1 sec
OVX 6W+E2 3W
0
E3
5 sec
5 min 1 sec
Figure 2. Differences of bladder activity (top traces) and EUS EMG activity (bottom traces in each set of records) among controls (SHAM), and rats with 4 (OVX 4W), 6 (OVX 6W) and 12-week (OVX 12W) OVX, and 6-week OVX plus 3-week E2 (OVX 6W⫹E2 3W). Recordings in A to E and A2 to E3 show same recordings at different time scales. A2 to E3, period during voiding in A to E but at faster time scale. Bottom traces of A2 to E3 show individual EUS EMG bursts to illustrate that SPs during voiding were shortened in OVX rats but normalized in OVX rats with E2 replacement.
activity after OVX are also due to an alteration in central mechanisms after a decrease in sex hormone levels. E2 treatment for 3 weeks starting 3 weeks after OVX reversed the changes in SP duration and VE. However, E2 treatment also increased micturition VT, which was not significantly changed by OVX. This suggests that large doses of E2 have an inhibitory
effect on the bladder or on the reflex pathways controlling micturition. In urethane anesthetized rats capsaicin sensitive, C-fiber bladder afferent nerves regulate bladder VT for initiating voiding. Thus, pretreatment with resiniferatoxin, which desensitizes Cfiber afferents, increases VT, similar to the effect of E2.19 Since estrogen receptors are expressed in afferent neurons20 –22 and estrogen treatment
EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
inhibits capsaicin evoked currents in cultured Cfiber type afferent neurons, it is possible that the effect of E2 on VT is due to suppression of the excitability of bladder C-fiber afferent pathways. Several central nervous system sites that regulate bladder and EUS function express estrogen receptors in normal animals or those with OVX, including 1) spinal interneurons and preganglionic neurons, 2) brainstem neurons projecting to spinal motoneurons and preganglionic neurons, and 3) a relay center in the brain stem (periaqueductal gray) involved in the micturition reflex.20 –24 Changes in the activity of neurons at these sites could underlie the effects of OVX or estrogen treatment. Gonadal hormones alter the expression of neurotransmitter receptors and the actions of neurotransmitters25–30 that are known to have a role in the central neural pathways controlling micturition. For example, estrogen receptors are co-expressed with receptors for the monoaminergic transmitters norepinephrine and serotonin in neurons at various sites in the central nervous system.26,28,30 Estrogen enhances the effects of norepinephrine mediated by ␣1-adrenoceptors.26,27 Depending on the pathway estrogen can enhance or inhibit the effects of serotonin mediated by the activation of 5-HT1A
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receptors.28,30 Agonists for ␣1 and 5-HT1A receptors enhance the reflex activity of the bladder and EUS in rats.8,10,12,14 The 5-HT1A agonist 8-OHDPAT increases EUS bursting and promotes VE in spinal cord injured rats with detrusor-sphincter dyssynergia.10 On the other hand, the 5-HT1A antagonist WAY-100635 shortens the SP of EUS EMG during bursting and decreases VE,14 an effect similar to the effect of OVX. In our experiments in rats with OVX E2 treatment also increased EUS bursting and VE, raising the possibility that E2 elicited its effect in part by enhancing monoaminergic (serotonin or norepinephrine) facilitatory control of EUS reflex pathways. In future studies it will be important to evaluate the interaction of estrogens and serotonergic drugs on LUT function.
CONCLUSIONS The major effect of chronic sex hormone deprivation on female rat LUT function was a decrease in bladder-sphincter coordination, which was evident as a decreased duration of sphincter relaxation during voiding, leading to decreased VE and urinary retention. The effect is attributable to a change in the central reflex pathways controlling EUS activity and it was reversed by E2 therapy.
REFERENCES 1. Rosenzweig BA, Bolina PS, Birch L et al: Location and concentration of estrogen, progesterone, and androgen receptors in the bladder and urethra of the rabbit. Neurourol Urodyn 1995; 14: 87. 2. Tincello DG, Taylor AH, Spurling SM et al: Receptor isoforms that mediate estrogen and progestagen action in the female lower urinary tract. J Urol 2009; 181: 1474. 3. Wolf H, Wandt H and Jonat W: Immunohistochemical evidence of estrogen and progesterone receptors in the female lower urinary tract and comparison with the vagina. Gynecol Obstet Invest 1991; 32: 227. 4. Fleischmann N, Christ G, Sclafani T et al: The effect of ovariectomy and long-term estrogen replacement on bladder structure and function in the rat. J Urol 2002; 168: 1265. 5. Robinson D and Cardozo LD: The role of estrogens in female lower urinary tract dysfunction. Urology 2003; 62: 45. 6. Batra S and Andersson KE: Oestrogen-induced changes in muscarinic receptor density and contractile responses in the female rabbit urinary bladder. Acta Physiol Scand 1989; 137: 135. 7. Yoshida J, Aikawa K, Yoshimura Y et al: The effects of ovariectomy and estrogen replacement on acetylcholine release from nerve fibres and passive stretch-induced acetylcholine release in
female rat bladder. Neurourol Urodyn 2007; 26: 1050.
tract dysfunction induced by chronic spinal cord injury in rats. Exp Neurol 2004; 187: 445.
8. Chang HY, Cheng CL, Chen JJ et al: Serotonergic drugs and spinal cord transections indicate that different spinal circuits are involved in external urethral sphincter activity in rats. Am J Physiol Renal Physiol 2007; 292: F1044.
14. Cheng CL and de Groat WC: Role of 5-HT1A receptors in control of lower urinary tract function in anesthetized rats. Am J Physiol Renal Physiol 2010; 298: F771.
9. Cheng CL, Chai CY and de Groat WC: Detrusorsphincter dyssynergia induced by cold stimulation of the urinary bladder of rats. Am J Physiol 1997; 272: R1271. 10. Dolber PC, Gu B, Zhang X et al: Activation of the external urethral sphincter central pattern generator by a 5-HT(1A) receptor agonist in rats with chronic spinal cord injury. Am J Physiol Regul Integr Comp Physiol 2007; 292: R1699. 11. Kruse MN, Belton AL and de Groat WC: Changes in bladder and external urethral sphincter function after spinal cord injury in the rat. Am J Physiol 1993; 264: R1157. 12. Chang HY, Cheng CL, Chen JJ et al: Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats. Am J Physiol Regul Integr Comp Physiol 2006; 291: R224. 13. Cheng CL and de Groat WC: The role of capsaicin-sensitive afferent fibers in the lower urinary
15. Peng CW, Chen JJ, Chang HY et al: External urethral sphincter activity in a rat model of pudendal nerve injury. Neurourol Urodyn 2006; 25: 388. 16. Kullmann FA, Limberg BJ, Artim DE et al: Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy. J Pharmacol Exp Ther 2009; 330: 704. 17. Diep N and Constantinou CE: Age dependent response to exogenous estrogen on micturition, contractility and cholinergic receptors of the rat bladder. Life Sci 1999; 64: PL279. 18. Streng T, Santti R, Andersson KE et al: The role of the rhabdosphincter in female rat voiding. BJU Int 2004; 94: 138. 19. Chuang YC, Fraser MO, Yu Y et al: Analysis of the afferent limb of the vesicovascular reflex using neurotoxins, resiniferatoxin and capsaicin. Am J Physiol Regul Integr Comp Physiol 2001; 281: R1302.
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20. Bennett HL, Gustafsson JA and Keast JR: Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder. Auton Neurosci 2003; 105: 90. 21. Papka RE and Mowa CN: Estrogen receptors in the spinal cord, sensory ganglia, and pelvic autonomic ganglia. Int Rev Cytol 2003; 231: 91. 22. VanderHorst VG, Meijer E and Holstege G: Estrogen receptor-alpha immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat. Neurosci Lett 2001; 298: 147. 23. VanderHorst VG, Meijer E, Schasfoort FC et al: Estrogen receptor-immunoreactive neurons in the lumbosacral cord projecting to the periaqueductal
gray in the ovariectomized female cat. Neurosci Lett 1997; 236: 25. 24. Williams SJ and Papka RE: Estrogen receptorimmunoreactive neurons are present in the female rat lumbosacral spinal cord. J Neurosci Res 1996; 46: 492. 25. Alfinito PD, Chen X, Mastroeni R et al: Estradiol increases catecholamine levels in the hypothalamus of ovariectomized rats during the darkphase. Eur J Pharmacol 2009; 616: 334. 26. Banie L, Lin G, Ning H et al: Effects of estrogen, raloxifene and levormeloxifene on alpha1A-adrenergic receptor expression. J Urol 2008; 180: 2241. 27. Lee AW, Kyrozis A, Chevaleyre V et al: Estradiol modulation of phenylephrine-induced excitatory
responses in ventromedial hypothalamic neurons of female rats. Proc Natl Acad Sci USA 2008; 105; 7333. 28. Rybaczyk LA, Bashaw MJ, Pathak DR et al: An overlooked connection. serotonergic mediation of estrogen-related physiology and pathology. BMC Women’s Health 2005; 5: 12. 29. Thompson AD, Angelotti T, Nag S et al: Sexspecific modulation of spinal nociception by alpha2-adrenoceptors. differential regulation by estrogen and testosterone. Neuroscience 2008; 153: 1268. 30. Xu H, Qin S, Carrasco GA et al: Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus. Neuroscience 2009; 158: 1599.
Abbreviations and Acronyms 5-HT1A ⫽ 5-hydroxytryptamine1A AP ⫽ active period BD ⫽ bursting duration CA ⫽ contraction amplitude CD ⫽ contraction duration CMG ⫽ cystometrogram E2 ⫽ estradiol EMG ⫽ electromyogram EUS ⫽ external urethral sphincter LUT ⫽ lower urinary tract OVX ⫽ ovariectomy PE ⫽ polyethylene RV ⫽ post-void residual volume SP ⫽ silent period TSP ⫽ total SP VE ⫽ voiding efficiency VT ⫽ volume threshold Submitted for publication September 22, 2010. Study received Taichung Veterans General Hospital institutional animal care and use committee approval. Supported by Grant NSC97-2314-B-075A-009MY3 from the National Science Foundation, Taiwan, Republic of China. * Correspondence: Division of Urology, Department of Surgery, Taichung Veterans General Hospital, 160, Section 3, Taichung-Kang Rd., Taichung, Taiwan, Republic of China (telephone: 886-42374-1215; FAX: 886-42359-3160; e-mail; [email protected]). † Financial interest and/or other relationship with Endo Pharmaceutical, Pfizer, Medtronic, Ethicon, Astellas, Eli Lilly and Takeda.
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Purpose: The postmenopausal hypoestrogen condition is associated with various lower urinary tract dysfunctions, including frequency, urgency, stress urinary incontinence and recurrent urinary infection. We determined whether hypoestrogen induced lower urinary tract dysfunction after ovariectomy is also associated with an alteration in external urethral sphincter activity. Materials and Methods: Bilateral ovariectomy was performed in female SpragueDawley® rats and sham operated rats served as controls. Transvesical cystometry and external urethral sphincter electromyogram activity were monitored 4, 6 and 12 weeks after sham operation or bilateral ovariectomy and at 6 weeks in bilaterally ovariectomized rats treated with estrogen. Results: The micturition reflex was elicited in sham operated and bilaterally ovariectomized, urethane anesthetized animals. Post-void residual urine increased and voiding efficiency decreased in rats with 4 to 12 weeks of bilateral ovariectomy. The silent period of external urethral sphincter electromyogram activity was shortened significantly and progressively at increased times after bilateral ovariectomy. These effects were prevented by estradiol treatment. Conclusions: As evidenced by shortening of the external urethral sphincter electromyogram silent period in ovariectomized rats, the disruption of coordination between the external urethral sphincter and the detrusor muscle could decrease urine outflow and in turn voiding efficiency. Estrogen replacement reverses these changes, suggesting that the central pathways responsible for detrusor-sphincter coordination are modulated by gonadal hormones. Key Words: urethra, ovariectomy, estrogens, menopause, urination disorders THE LUT and genital organs, which share a common embryological origin, express estrogen and progesterone receptors, and show physiological responses to these hormones.1–3 Postmenopausal hypoestrogen status elicits anatomical and physiological changes in LUT4 and may be associated with various LUT dysfunctions, including frequency, urgency, urge incontinence, stress urinary incontinence, detrusor overactivity and recurrent urinary tract infections.5 The influence of gonadal hormones on
LUT function is likely mediated via multiple effects on neural control, vascular supply, and detrusor muscle cell size and number as well as by connective tissue density and distribution.4 –7 Most groups have focused on functional and morphological changes in the bladder and urethral smooth muscle in the hypoestrogen condition after menopause or OVX in human and animal models, respectively.2,4,6,7 We explored the effect of OVX on EUS activity and its coordination with the
0022-5347/11/1861-0334/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 334-340, July 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.03.035
AND
RESEARCH, INC.
EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
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Bilateral OVX was performed via a midline abdominal approach using isoflurane anesthesia and aseptic techniques. In sham operated controls the 2 ovaries were visualized but not removed. The animals were treated with antibiotic (ampicillin 150 mg/kg intramuscularly daily) for 2 to 3 days to prevent infection after surgery.
where EUS muscle fibers were identified. The recorded EUS EMG was attributable to striated muscle since the activity was eliminated after neuromuscular blockade with pancuronium bromide (Organon, Oss, The Netherlands).9 A PE 60 tube with a 1.0 mm inner diameter and 1.5 mm outer diameter was inserted in the bladder lumen. The bladder end of the PE tube was heated to create a collar and passed through a small incision at the apex of the bladder dome. A suture was tightened around the collar of the tube. The abdominal wall was then closed with nylon suture. The PE tube was in turn connected via a 3-way stopcock to an infusion pump and a pressure transducer. The system was then filled with physiological saline. Urodynamic examination usually began 3 to 4 hours after anesthesia induction. After the bladder was emptied transvesical cystometry was done at an infusion rate of 0.123 ml per minute with physiological saline at room temperature. The urethra was open and fluid could be evacuated during micturition. The infusion pump was turned off after induction of a voiding contraction. RV was measured by empting the bladder by pressure on the abdominal wall. Various parameters were measured, including VT (saline volume sufficient to induce bladder contraction exceeding 15 cm H2O), CA (maximal intravesical pressure during voiding), CD, RV (saline volume withdrawn through the intravesical catheter after voiding) and VE, expressed as a percent using the formula, VE ⫽ voided volume (VT ⫺ RV)/VT ⫻ 100. Investigators performing EUS EMG activity analysis were blinded to rat status. As previously described,13 various EUS EMG parameters were measured, including average BD, SP and AP (fig. 1), TSP during each void and the ratio of BD to CD, expressed as a percent. Bursting was measured during the time when EMG was completely quiescent between bursts (fig. 1), which occurred when intravesical pressure began to decrease during voiding. Before this time some phasic EMG activity was detected but an SP was not obvious (fig. 1). At least 5 transvesical CMGs were done per animal. All parameters were calculated with AcqKnowledge™. Calculated data were compiled in spreadsheets using Excel®. EUS EMG activity was displayed on a storage oscilloscope and a paper recorder along with bladder pressure.
E2 Treatment
Statistical Analysis
The rats were randomly assigned to hormone replacement with 17-E2 (0.05 mg as a 21-day time release E2 pellet) (Innovative Research of America, Sarasota, Florida). The pellet was implanted subcutaneously at the mid scapular level 3 weeks after OVX.
Results are shown as the mean ⫾ SE. Data were tested for normal distribution using the Shapiro-Wilk test and found to be not normally distributed (p ⬍0.01). Thus, associations between sham operated and OVX rats were assessed by the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test with the p value adjusted for the 4 comparisons using the Bonferroni correction. Differences were considered statistically significant at p ⬍0.05.
detrusor muscle during voiding in anesthetized female rats. In previous experiments at various laboratories groups reported that EUS EMG in rats shows tonic activity during bladder filling and bursting activity during voiding.8 –12 Bursting can be divided into APs and SPs, and analyzed using various parameters, including the duration and interval of these periods and of total BD.13 The pattern of EUS EMG activity can be altered by drugs8,10,12,14 and pathological conditions such as spinal cord or pudendal nerve injury while changes in EUS EMG activity correlate with changes in voiding function.8,10,13,15 The current experiments revealed that OVX also produces prominent changes in EUS EMG activity that are linked to decreased VE.
MATERIALS AND METHODS Experimental Protocols Adult female Sprague-Dawley rats were housed individually in a room controlled for temperature, humidity and lighting with lights on from 7:00 a.m. to 7:00 p.m. Rats had free access to food and water. A total of 76 female Sprague-Dawley rats weighing 230 to 320 gm were used in this study. The animals were divided into 7 groups. Groups 1 to 3 underwent sham operation and were studied 4 (11 rats), 6 (12) and 12 (11) weeks postoperatively, respectively. Groups 4 to 6 underwent OVX and were studied at 4 (11 rats), 6 (11) and 12 (10) weeks postoperatively, respectively. Group 7 (10 rats) underwent bilateral OVX, and was treated with 17-E2 replacement for 3 weeks starting 3 weeks after OVX and studied 6 weeks after OVX. The Taichung Veterans General Hospital institutional animal care and use committee approved the study protocol.
OVX Surgery
Physiological Investigation Experiments were done using urethane anesthesia (1.2 gm/kg subcutaneously). The femoral vein was catheterized for fluid administration. Body temperature was maintained between 36C and 38C with a heating lamp. The bladder was exposed via a midline abdominal incision. The rostral half of the pubic symphysis was removed to expose the mid urethra and the EUS. Two fine insulated silver wire electrodes 0.05 mm in diameter with exposed tips were inserted into the lateral sides of the mid urethra,
RESULTS Uterus and Body Weight The mean body weight of rats with 4 to 12-week OVX was significantly larger than that of time matched, sham operated rats 8% to 21%, (p ⬍0.05,
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EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
Figure 1. Bladder (A, C, E and G) and EUS EMG (B, D, F and H) activity recorded during continuous transvesical infusion CMG in anesthetized rat. Reflex bladder contraction, indicated by large, abrupt bladder pressure increase, was accompanied by large amplitude EUS EMG activity (A and B). Vertical calibration represents intravesical pressure in cm H2O (A). Arrow indicates start of saline infusion (A). Asterisk indicates C and D. Reflex bladder contraction recording at more rapid time scale (C and D). Bracket (C) indicates E and F. Tonic EUS EMG activity preceded large intravesical pressure increase and shifted to bursting pattern at bladder contraction peak before voiding onset (E and F). Bracket (E) indicates G and H. Recordings at more rapid time scale show individual EUS EMG bursts composed of APs and SPs (brackets), and small intravesical pressure fluctuations accompanying each burst (G and H).
table 1). The uterus and fallopian tubes appeared atrophied in rats with OVX. The uterine weight of rats with 4 to 12-week OVX was significantly lower than of sham operated rats (18% to 26% of control values, table 1). OVX Effects Bladder activity during transvesical CMG. With the urethral outlet open to allow intravesical fluid to be evacuated during voiding, various parameters of LUT activity and EUS EMG were assessed during slow saline infusion, transvesical CMG. The micturition reflex was elicited in all controls and rats with OVX at different postoperative intervals (4 to 12 weeks). However, some bladder activity parameters changed at different times after OVX. VT, CA and CD did not change 4 to 12 weeks after OVX but RV increased significantly from 0.27 to 0.38 ml starting 4 weeks after OVX (table 2). VE decreased progressively from 57.7% to 43.9% after OVX despite unchanged maximal voiding pressure (table 2).
EUS EMG activity. During continuous infusion CMG before the onset of voiding 2 types of EUS EMG activity were detected. Most rats showed consistent, low amplitude, tonic EUS EMG activity during the filling phase. In some animals this tonic activity increased gradually as infusion volume approached the micturition VT. During bladder contraction the EUS EMG activity markedly increased, consisting of an initial period of tonic activity interspersed with phasic activity in some rats, followed by a bursting pattern of activity characterized by clusters of high frequency spikes (AP) separated by quiescent periods (SP). SP decreased significantly and progressively 21% to 35% at increased times after OVX (table 2 and fig. 2). TSP was significantly increased 4 and 6 weeks after OVX but not at 12 weeks (table 2). There was no difference in AP duration between rats with OVX and controls at 4 weeks but AP duration significantly increased 6 and 12 weeks after OVX (table 2 and fig. 2). BD and the ratio of BD to CD were decreased 13% to 16% 4 and 6 weeks after OVX, respectively, but these changes were not statistically significant (table 2). Effect of Post-OVX E2 Replacement on Bladder Activity Transvesical CMG done after 3 weeks of E2 treatment (0.05 mg as a 21-day time release E2 pellet), which started 3 weeks after OVX, revealed that the VT increased (85%) in E2 treated animals compared to that in rats with 6-week OVX, although OVX did not decrease bladder VT (table 2). The change in VE that occurred after OVX was completely reversed by E2 treatment (table 2). Also, SP, BD and TSP were significantly increased by E2 treatment (table 2 and fig. 2).
Table 1. Effect of OVX and E2 replacement on body and uterine weight Mean ⫾ SE Wt
4 Wks: Control OVX p Value 6 Wks: Control OVX p Value OVX ⫹ 3-wk E2 p Value 12 Wks: Control OVX p Value
No. Rats
Body (gm)
Uterus (mg)
8 9
298.5 ⫾ 16.9 358.1 ⫾ 35.2 0.000*
476.0 ⫾ 98.6 124.3 ⫾ 16.0 0.016*
9 9
335.8 ⫾ 23.8 363.3 ⫾ 27.4 0.002* 326.0 ⫾ 12.2 0.000*
542.0 ⫾ 82.9 140.9 ⫾ 28.1 0.000* 508.5 ⫾ 152.0 0.000*
328.6 ⫾ 22.0 400.7 ⫾ 34.5 0.000*
561.9 ⫾ 93.2 102.5 ⫾ 12.4 0.000*
8
9 8
* Mann-Whitney U test with Bonferroni correction p ⬍0.05.
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0.120 ⫾ 0.010 0.067 ⫾ 0.008 5.63 ⫾ 1.80 3.62 ⫾ 1.18 25.17 ⫾ 7.28
0.093 ⫾ 0.074 ⫾ 4.69 ⫾ 2.65 ⫾ 21.18 ⫾
0.017 0.007 2.22 1.49 7.94
0.002* 0.034* 0.171 0.040* 0.365
0.105 ⫾ 0.028 0.068 ⫾ 0.007 7.03 ⫾ 4.83 4.42 ⫾ 3.75 27.34 ⫾ 10.14
0.349 0.085 0.043* 0.020* 0.051
0.111 ⫾ 0.013 0.068 ⫾ 0.004 5.75 ⫾ 2.96 3.55 ⫾ 1.79 23.52 ⫾ 7.70
0.084 ⫾ 0.079 ⫾ 6.22 ⫾ 3.22 ⫾ 23.17 ⫾
0.012 0.007 4.16 2.26 9.80
0.000* 0.004* 0.705 0.282 0.426
DISCUSSION
* Mann-Whitney U test with Bonferroni correction p ⬍0.05.
0.021* 0.332 0.151 0.047* 0.171 0.022 0.010 2.13 1.17 6.88 0.096 ⫾ 0.072 ⫾ 5.39 ⫾ 3.15 ⫾ 22.98 ⫾ 0.127 ⫾ 0.017 0.066 ⫾ 0.004 6.19 ⫾ 1.26 4.12 ⫾ 0.88 28.09 ⫾ 7.42
0.809 0.654 0.863 0.008* 0.003* ⫾ 0.27 ⫾ 5.76 ⫾ 0.16 ⫾ 0.22 ⫾ 16.92 0.67 36.10 0.43 0.38 43.85 0.49 33.18 0.38 0.08 83.23
No. rats Cystometry: VT (ml) CA (cm H2O) CD (mins) RV (ml) % VE EUS EMG: SP (secs) AP (secs) BD (secs) TSP (secs) % BD/CD
11
⫾ 0.22 ⫾ 5.54 ⫾ 0.05 ⫾ 0.06 ⫾ 8.34
11
0.60 38.68 0.40 0.27 57.74
⫾ 0.22 ⫾ 2.59 ⫾ 0.11 ⫾ 0.19 ⫾ 21.81
0.243 0.016* 0.847 0.007* 0.004*
0.50 32.76 0.38 0.12 75.15
12
⫾ 0.12 ⫾ 4.48 ⫾ 0.09 ⫾ 0.04 ⫾ 8.34
11
0.54 39.09 0.36 0.29 48.64
⫾ 0.20 ⫾ 3.95 ⫾ 0.06 ⫾ 0.15 ⫾ 11.69
0.438 0.016* 0.438 0.002* 0.000*
1.00 31.61 0.41 0.26 74.19
10
⫾ 0.34 ⫾ 6.03 ⫾ 0.10 ⫾ 0.12 ⫾ 10.38
0.004* 0.015* 0.349 0.809 0.001*
0.63 34.60 0.40 0.20 67.91
11
⫾ 0.13 ⫾ 6.13 ⫾ 0.09 ⫾ 0.05 ⫾ 4.62
10
Mean ⫾ SE OVX p Value Mean ⫾ SE OVX Mean ⫾ SE Control p Value Mean ⫾ SE OVX Mean ⫾ SE Control
4 Wks
Table 2. Effect of OVX and E2 replacement on cystometric parameters and EUS EMG activity
6 Wks
Mean ⫾ SE OVX ⫹ 3-Wk E2
p Value
Mean ⫾ SE Control
12 Wks
p Value
EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
The current experiments reveal that rats with OVX showed several voiding abnormalities, including 1) increased RV, 2) decreased VE and 3) an alteration in EUS EMG bursting activity, characterized primarily by a decreased SP duration during micturition. These observations indicate that coordination between bladder and EUS is disrupted by OVX, leading to a decrease in the periods of EUS relaxation, which in turn decreases the bladder emptying efficiency. These effects were reversed by E2 treatment. Previous studies in rats indicated that OVX increased voiding frequency,7,16 suggesting that a decrease in gonadal hormone levels increases the activity of bladder smooth muscle or the neural pathways controlling the bladder. One study also showed that OVX induced an age and time dependent change in urodynamic parameters, including an increase in urethral opening pressure and maximal voiding pressure as well as a decrease in maximal flow rate and voided volume.17 These changes were reversed by treatment with exogenous estrogen for 4 weeks. Our experiments in rats with OVX did not reveal a change in the VT to initiate micturition or in peak intravesical pressure during micturition. However, they revealed decreased VE associated with a decrease in the duration of EUS SPs and TSPs. The change in SP occurred at the earliest time tested after OVX (4 weeks) and increased in magnitude at longer times (6 to 12 weeks). EUS bursting activity reflects the rhythmic opening and closing of the outlet to produce a pulsatile flow of urine, which is common in rodents. Since bursting represents the period of flow through the urethra18 and SP represents the period of maximal urethral relaxation, it is reasonable to conclude that changes in EUS neural control contribute to the inefficient voiding and urinary retention seen in the current experiments. They also likely account for the decrease in the maximal urine flow rate reported by Diep and Constantinou in rats with OVX.17 On the other hand, it is uncertain whether the changes in bladder function after OVX that were reported by others contribute to decreased VE.6,7 OVX significantly increased body weight, an effect that may have increased urine production and produced the small but not significant increase in bladder VT. However, it is unlikely that the change in body weight contributed to the putative neurally mediated changes in EUS EMG activity or VE. SP shortening and decreased BD/CD were detected in chronic spinal cord transected rats, which show detrusor-sphincter dyssynergia, decreased VE and increased RV.13 In spinal cord transected rats the change in EUS EMG activity is clearly due to a central nervous system abnormality. By analogy it seems reasonable to propose that the changes in EUS EMG
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EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
A cmH2O 50
A2
A1 SHAM
0
5 min
B cmH2O 50
B1
A3
B2
5 sec
1 sec
OVX 4W
0
5 min
C cmH2O 50
B3
5 sec
1 sec
C1 OVX 6W
C2
0
5 min
D cmH2O 50
D1
C3
D2
5 sec
1 sec
OVX 12W
0
D3
5 sec
5 min
E cmH2O 50
E1
E2
1 sec
OVX 6W+E2 3W
0
E3
5 sec
5 min 1 sec
Figure 2. Differences of bladder activity (top traces) and EUS EMG activity (bottom traces in each set of records) among controls (SHAM), and rats with 4 (OVX 4W), 6 (OVX 6W) and 12-week (OVX 12W) OVX, and 6-week OVX plus 3-week E2 (OVX 6W⫹E2 3W). Recordings in A to E and A2 to E3 show same recordings at different time scales. A2 to E3, period during voiding in A to E but at faster time scale. Bottom traces of A2 to E3 show individual EUS EMG bursts to illustrate that SPs during voiding were shortened in OVX rats but normalized in OVX rats with E2 replacement.
activity after OVX are also due to an alteration in central mechanisms after a decrease in sex hormone levels. E2 treatment for 3 weeks starting 3 weeks after OVX reversed the changes in SP duration and VE. However, E2 treatment also increased micturition VT, which was not significantly changed by OVX. This suggests that large doses of E2 have an inhibitory
effect on the bladder or on the reflex pathways controlling micturition. In urethane anesthetized rats capsaicin sensitive, C-fiber bladder afferent nerves regulate bladder VT for initiating voiding. Thus, pretreatment with resiniferatoxin, which desensitizes Cfiber afferents, increases VT, similar to the effect of E2.19 Since estrogen receptors are expressed in afferent neurons20 –22 and estrogen treatment
EFFECT OF OVARIECTOMY ON EXTERNAL URETHRAL SPHINCTER ACTIVITY
inhibits capsaicin evoked currents in cultured Cfiber type afferent neurons, it is possible that the effect of E2 on VT is due to suppression of the excitability of bladder C-fiber afferent pathways. Several central nervous system sites that regulate bladder and EUS function express estrogen receptors in normal animals or those with OVX, including 1) spinal interneurons and preganglionic neurons, 2) brainstem neurons projecting to spinal motoneurons and preganglionic neurons, and 3) a relay center in the brain stem (periaqueductal gray) involved in the micturition reflex.20 –24 Changes in the activity of neurons at these sites could underlie the effects of OVX or estrogen treatment. Gonadal hormones alter the expression of neurotransmitter receptors and the actions of neurotransmitters25–30 that are known to have a role in the central neural pathways controlling micturition. For example, estrogen receptors are co-expressed with receptors for the monoaminergic transmitters norepinephrine and serotonin in neurons at various sites in the central nervous system.26,28,30 Estrogen enhances the effects of norepinephrine mediated by ␣1-adrenoceptors.26,27 Depending on the pathway estrogen can enhance or inhibit the effects of serotonin mediated by the activation of 5-HT1A
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receptors.28,30 Agonists for ␣1 and 5-HT1A receptors enhance the reflex activity of the bladder and EUS in rats.8,10,12,14 The 5-HT1A agonist 8-OHDPAT increases EUS bursting and promotes VE in spinal cord injured rats with detrusor-sphincter dyssynergia.10 On the other hand, the 5-HT1A antagonist WAY-100635 shortens the SP of EUS EMG during bursting and decreases VE,14 an effect similar to the effect of OVX. In our experiments in rats with OVX E2 treatment also increased EUS bursting and VE, raising the possibility that E2 elicited its effect in part by enhancing monoaminergic (serotonin or norepinephrine) facilitatory control of EUS reflex pathways. In future studies it will be important to evaluate the interaction of estrogens and serotonergic drugs on LUT function.
CONCLUSIONS The major effect of chronic sex hormone deprivation on female rat LUT function was a decrease in bladder-sphincter coordination, which was evident as a decreased duration of sphincter relaxation during voiding, leading to decreased VE and urinary retention. The effect is attributable to a change in the central reflex pathways controlling EUS activity and it was reversed by E2 therapy.
REFERENCES 1. Rosenzweig BA, Bolina PS, Birch L et al: Location and concentration of estrogen, progesterone, and androgen receptors in the bladder and urethra of the rabbit. Neurourol Urodyn 1995; 14: 87. 2. Tincello DG, Taylor AH, Spurling SM et al: Receptor isoforms that mediate estrogen and progestagen action in the female lower urinary tract. J Urol 2009; 181: 1474. 3. Wolf H, Wandt H and Jonat W: Immunohistochemical evidence of estrogen and progesterone receptors in the female lower urinary tract and comparison with the vagina. Gynecol Obstet Invest 1991; 32: 227. 4. Fleischmann N, Christ G, Sclafani T et al: The effect of ovariectomy and long-term estrogen replacement on bladder structure and function in the rat. J Urol 2002; 168: 1265. 5. Robinson D and Cardozo LD: The role of estrogens in female lower urinary tract dysfunction. Urology 2003; 62: 45. 6. Batra S and Andersson KE: Oestrogen-induced changes in muscarinic receptor density and contractile responses in the female rabbit urinary bladder. Acta Physiol Scand 1989; 137: 135. 7. Yoshida J, Aikawa K, Yoshimura Y et al: The effects of ovariectomy and estrogen replacement on acetylcholine release from nerve fibres and passive stretch-induced acetylcholine release in
female rat bladder. Neurourol Urodyn 2007; 26: 1050.
tract dysfunction induced by chronic spinal cord injury in rats. Exp Neurol 2004; 187: 445.
8. Chang HY, Cheng CL, Chen JJ et al: Serotonergic drugs and spinal cord transections indicate that different spinal circuits are involved in external urethral sphincter activity in rats. Am J Physiol Renal Physiol 2007; 292: F1044.
14. Cheng CL and de Groat WC: Role of 5-HT1A receptors in control of lower urinary tract function in anesthetized rats. Am J Physiol Renal Physiol 2010; 298: F771.
9. Cheng CL, Chai CY and de Groat WC: Detrusorsphincter dyssynergia induced by cold stimulation of the urinary bladder of rats. Am J Physiol 1997; 272: R1271. 10. Dolber PC, Gu B, Zhang X et al: Activation of the external urethral sphincter central pattern generator by a 5-HT(1A) receptor agonist in rats with chronic spinal cord injury. Am J Physiol Regul Integr Comp Physiol 2007; 292: R1699. 11. Kruse MN, Belton AL and de Groat WC: Changes in bladder and external urethral sphincter function after spinal cord injury in the rat. Am J Physiol 1993; 264: R1157. 12. Chang HY, Cheng CL, Chen JJ et al: Roles of glutamatergic and serotonergic mechanisms in reflex control of the external urethral sphincter in urethane-anesthetized female rats. Am J Physiol Regul Integr Comp Physiol 2006; 291: R224. 13. Cheng CL and de Groat WC: The role of capsaicin-sensitive afferent fibers in the lower urinary
15. Peng CW, Chen JJ, Chang HY et al: External urethral sphincter activity in a rat model of pudendal nerve injury. Neurourol Urodyn 2006; 25: 388. 16. Kullmann FA, Limberg BJ, Artim DE et al: Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy. J Pharmacol Exp Ther 2009; 330: 704. 17. Diep N and Constantinou CE: Age dependent response to exogenous estrogen on micturition, contractility and cholinergic receptors of the rat bladder. Life Sci 1999; 64: PL279. 18. Streng T, Santti R, Andersson KE et al: The role of the rhabdosphincter in female rat voiding. BJU Int 2004; 94: 138. 19. Chuang YC, Fraser MO, Yu Y et al: Analysis of the afferent limb of the vesicovascular reflex using neurotoxins, resiniferatoxin and capsaicin. Am J Physiol Regul Integr Comp Physiol 2001; 281: R1302.
340
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20. Bennett HL, Gustafsson JA and Keast JR: Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder. Auton Neurosci 2003; 105: 90. 21. Papka RE and Mowa CN: Estrogen receptors in the spinal cord, sensory ganglia, and pelvic autonomic ganglia. Int Rev Cytol 2003; 231: 91. 22. VanderHorst VG, Meijer E and Holstege G: Estrogen receptor-alpha immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat. Neurosci Lett 2001; 298: 147. 23. VanderHorst VG, Meijer E, Schasfoort FC et al: Estrogen receptor-immunoreactive neurons in the lumbosacral cord projecting to the periaqueductal
gray in the ovariectomized female cat. Neurosci Lett 1997; 236: 25. 24. Williams SJ and Papka RE: Estrogen receptorimmunoreactive neurons are present in the female rat lumbosacral spinal cord. J Neurosci Res 1996; 46: 492. 25. Alfinito PD, Chen X, Mastroeni R et al: Estradiol increases catecholamine levels in the hypothalamus of ovariectomized rats during the darkphase. Eur J Pharmacol 2009; 616: 334. 26. Banie L, Lin G, Ning H et al: Effects of estrogen, raloxifene and levormeloxifene on alpha1A-adrenergic receptor expression. J Urol 2008; 180: 2241. 27. Lee AW, Kyrozis A, Chevaleyre V et al: Estradiol modulation of phenylephrine-induced excitatory
responses in ventromedial hypothalamic neurons of female rats. Proc Natl Acad Sci USA 2008; 105; 7333. 28. Rybaczyk LA, Bashaw MJ, Pathak DR et al: An overlooked connection. serotonergic mediation of estrogen-related physiology and pathology. BMC Women’s Health 2005; 5: 12. 29. Thompson AD, Angelotti T, Nag S et al: Sexspecific modulation of spinal nociception by alpha2-adrenoceptors. differential regulation by estrogen and testosterone. Neuroscience 2008; 153: 1268. 30. Xu H, Qin S, Carrasco GA et al: Extra-nuclear estrogen receptor GPR30 regulates serotonin function in rat hypothalamus. Neuroscience 2009; 158: 1599.