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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS
Departments of 1Urology and 3Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and 2Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka 532-8686, Japan
Submitted 8 January 2003 ; accepted in final form 12 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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80% by bilateral
transection of the pudendal nerves and the nerves to the iliococcygeous and
pubococcygeous muscles but not by transection of the visceral branches of the
pelvic nerves and hypogastric nerves. The sneeze leak point pressure was also
measured to investigate the role of active urethral closure mechanisms in
maintaining total urethral resistance against sneeze-induced urinary
incontinence. In sham-operated rats, no urinary leakage was observed during
sneeze, which produced an increase of intravesical pressure up to 37 ±
2.2 cmH2O. However, in nerve-transected rats urinary leakage was
observed when the intravesical pressure during sneezing exceeded 16.3 ±
2.1 cmH2O. These results indicate that during sneezing, pressure
increases elicited by reflex contractions of external urethral sphincter and
pelvic floor muscles occur in the middle portion of the urethra. These
reflexes in addition to passive transmission of increased abdominal pressure
significantly contribute to urinary continence mechanisms under a
sneeze-induced stress condition. urethral resistance; active urethral closure; sneeze leak point pressure
Urinary continence is maintained during elevation of abdominal pressure by multiple mechanisms. The passive closure of the urethra by the transmission of increased abdominal pressure to the urethra has been considered to play an important role in urinary continence; in addition, under stress conditions neurally mediated urethral closure has also been proposed based on various clinical findings, including the following: 1) the rise in urethral pressure starts before cough transmission (27, 28, 33), 2) the increase in urethral pressure during coughing exceeds the increase in bladder pressure (5, 6, 27, 28), and 3) urethral closure forces during coughing were significantly reduced by bilateral pudendal nerve blockade (26).
It has recently been reported that in SUI patients the incidence of intrinsic sphincter deficiency, characterized by a malfunction of the urethral sphincter mechanism resulting in the low-pressure urethra, was greater than previously thought (13). Therefore, it seems important to study the active urinary closure mechanism under stress conditions. In animal studies, the details of urethral closure mechanisms during elevation of abdominal pressure have not been studied except in dogs, in which two components (passive and active) of urethral closure mechanisms were detected (10, 30, 31).
The aim of this study was to clarify urinary continence mechanisms during the elevation of abdominal pressure induced by sneezing in rats. Sneeze, which is a highly coordinated reflex evoked by irritation of nasal mucosa, is designed to remove irritants and clean the airway. Previous studies have used this reflex to induce urinary leakage in a rat model of SUI induced by intravaginal balloon inflation (15, 22). It is hoped that the development of a reproducible animal model of SUI will lead to better understanding of pathophysiology of this condition and to develop new treatments for SUI.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Forty-eight adult female rats of Sprague-Dawley strain weighing 212–293 g were studied according to experimental protocols approved by University of Pittsburgh Institutional Animal Care and Use Committee.
Experiment 1: Bladder and Urethral Responses During Sneezing
Five rats were anesthetized by halothane (Halocarbon Laboratories, River Edge, NJ) inhalation, and a polyethylene catheter (PE-50, Clay Adams, Parsippany, NJ) was inserted into a carotid artery for recording arterial blood pressure. The urinary bladder was exposed through an abdominal incision, and both ureters were cut and their distal ends were ligated. A 3.5-Fr. size nylon catheter with a side-mounted microtip transducer located 1 mm from the catheter tip (SPR-524, Millar Instruments, Houston, TX) was inserted into the bladder from the dome. The abdomen was then closed with sutures.
After the surgery, halothane anesthesia was turned off and replaced with
urethane anesthesia (0.72 g/kg sc, Sigma, St. Louis, MO). Additional doses of
anesthetic were then administered intravenously as required during the
experiments. Another microtip transducer catheter was inserted into the
urethra from the urethral orifice, with its side-mounted sensor facing the
inner urethral surface in the 3 o'clock position because a measurement in a
lateral orientation corresponds most closely to the urethral pressure
(1). Although transurethral
catheters might affect bladder and urethral functions by dilating the urethra
and activating afferent nerves, microtip transducer catheters were used
because there are currently no noninvasive methods available for measuring
local responses in a restricted portion of the urethra. The urethral length in
female rats, which was determined by the length of microtip transducer
catheters inserted into the urethra from the urethral orifice, was 20
mm.
In preliminary experiments, the different characteristics of urethral response during sneezing were observed at proximal, middle, and distal portions of the urethra (15–20, 10–15, and 0–10 mm from the urethral orifice, respectively) by changing the position of the microtip transducer catheter in the same rats. The length of catheter inserted into the urethra from the urethral orifice was measured before and after inducing the sneeze reflex to confirm that the position of transducer was not moved during the sneeze reflex. The bladder was emptied before start of the experiment so as to measure response under standardized conditions. This procedure was necessary for recording pressures independently using microtip transducer catheters in the bladder and urethra and also allowed us to focus on the sneeze-induced urethral reflex without influences of possible bladder-to-urethral reflexes induced by bladder distension. A rat whisker was cut and inserted into the nostrils to induce the sneeze reflex. Microtip transducer catheters were connected to an amplifier (Transbridge 4M, World Precision Instruments, Sarasota, FL), and bladder (Ves) and urethral (Ura) responses, and their difference (Ura-Ves difference) during sneezing were recorded using data acquisition software (Chart, ADInstruments, Castle Hill, NSW, Australia) on a computer system equipped with an analog-to-digital converter (Power lab, ADInstruments). Sneeze-induced bladder and urethral responses measured by microtip transducer catheters, which strictly speaking correspond to the force per unit area exerted by the wall of the organ on the catheter-mounted sensor, are approximately equal to changes in the bladder and urethral pressures and were expressed in centimeters H2O (1, 9). As the magnitude of the sneeze reflex varied in each episode, at least 10 sneeze reflexes were induced in each test. In three of five rats, the sneeze reflex was also induced after opening of the abdomen. In addition, simultaneous recordings of rectal responses using microtip transducer catheters were performed in three other rats to examine whether sneeze-induced pressure increases in the abdomen produce similar responses in the bladder and rectum. We also confirmed in preliminary experiments (n = 3) that sneeze-induced rectal responses were similar before and after opening and subsequent closing of the abdomen with sutures. Thus it seems that when the abdomen was closed with the method used in this study, it was reliably airtight.
Experiment 2: Effects of Bilateral Transection of Nerves Innervating Urethra on Urethral Responses During Sneezing
Twenty-eight rats were used to evaluate the contribution to urethral closure of each nerve innervating the urethra. Under halothane anesthesia, pudendal nerves (n = 6), nerves to iliococcygeous and pubococcygeous muscles [somatomotor branch of pelvic nerve as described in a previous report (16); n = 7], or both pudendal nerves and nerves to iliococcygeous and pubococcygeous muscles (n = 5) were transected bilaterally near internal iliac vessels according to the method of Manzo et al. (16). In three rats, hypogastric nerves and visceral branches of the pelvic nerves were transected bilaterally near the major pelvic ganglia. In seven rats, a sham operation was performed. The bladder and middle urethral responses during sneezing were recorded as described in experiment 1.
Experiment 3: Effects of Bilateral Transection of Pudendal Nerves on EMG Recordings of External Urethral Sphincter
In another series of experiments, the change in external urethral sphincter (EUS)-EMG activity during sneezing was evaluated (n = 7). In three of seven rats, pudendal nerves were transected bilaterally under halothane anesthesia. A pair of wire electrodes (1512F, Life-Tech, Stafford, TX) was inserted into the EUS under urethane anesthesia and connected to an amplifier (Grass P511, Astro-Med, West Warwick, RI). The sneeze reflex was induced as described above, and changes in EMG recordings of EUS were recorded on a computer system equipped with an analog-to-digital converter using the data acquisition software. Recorded EMG activity was integrated for each 1 s before and after start of the sneeze reflex, and the increase in integrated EMG value during sneezing was calculated. In addition, EMG activity in anal sphincter muscles was also evaluated in three other rats to examine whether similar reflex activities during sneezing occur in the anal sphincter.
Experiment 4: Effects on Sneeze Leak Point Pressure of Bilateral Transection of Pudendal Nerves and Nerves to Iliococcygeous and Pubococcygeous Muscles
Eight rats were anesthetized by halothane inhalation, and the urinary bladder was exposed through an abdominal incision. A catheter (PE-50) connected to a pressure transducer (BLPR, World Precision Instruments) was inserted into the bladder from the dome for recording the intravesical pressure. The visceral branches of pelvic nerves were cut bilaterally near internal iliac vessels so that reflex bladder contractions as well as the bladder-to-urethral reflex induced by bladder distension were prevented. The eight rats were then divided into two groups (each 4 rats) with or without bilateral transection of both the pudendal nerves and the nerves to the iliococcygeous and pubococcygeous muscles. The abdomen was then closed with sutures.
After the surgery, the anesthesia was switched to urethane anesthesia. After the bladder was emptied, 0.4 ml of saline solution containing Evans blue (100 µg/ml; Sigma) was injected into the bladder. The sneeze reflex was induced as described above at least 50 times while changes were recorded in intravesical pressure to examine whether urinary leakage from the urethral orifice was induced. The maximal intravesical pressure was measured during each sneeze event, and the lowest pressure value that induced fluid leakage from the urethral orifice was defined as the sneeze leak point pressure.
Statistical Analysis
Data are expressed as means ± SE. In experiments 1–3, at least 10 sneeze-evoked responses were recorded in one series under the same experimental conditions, and the values were averaged in each rat. The mean ± SE in a group of animals was then calculated from the averaged value in each rat except for Fig. 5, where the method of analysis was different, as described in RESULTS. To analyze the statistical difference in responses of proximal, middle, and distal urethra during sneezing (experiment 1, Table 1) Dunnett's test was used. For the analysis of statistical differences in the mean values among nerve-transected groups (experiment 2, Table 2), Dunnett's test or Student's t-test was used. The statistical difference of the mean change in EMG activity during sneezing (experiment 3) was analyzed between the sham-operated and the pudendal nerve-transected groups by a Student's t-test. Probability values (P) <0.05 were considered to be significant. In experiment 4, at least 50 sneeze reflexes were induced, and the following parameters (sneeze leak point pressure, maximal intravesical pressure without fluid leakage among all sneeze events, average of maximal intravesical pressure in each sneeze event, and incidence of fluid leakage) were calculated in each rat. The mean ± SE was then obtained in each group of animals.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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When the sneeze reflex was induced by stimulation of nostril inner surface with a rat whisker, blood pressure consistently decreased after induction of a sneeze (Figs. 1 and 2). Bladder and urethral responses in the proximal, middle, and distal parts during sneezing were measured using microtip transducer catheters under an abdomen-closed condition (n = 5), and then, in three of five rats, the abdomen was opened to eliminate the increase in abdominal pressure during sneezing, and responses induced by sneezing were then recorded.
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Bladder. The readings measured by a microtip transducer catheter in the bladder increased during sneezing and returned to the baseline within 0.15 s under an abdomen-closed condition (Figs. 1 and 3). When rectal responses were measured using a microtip transducer catheter, a similar shape of short-lasting pressure rises was observed simultaneously in the bladder and rectum during sneezing, indicating that these short-lasting sneeze-induced pressure rises in the bladder are not movement artifacts. In addition, long-lasting (<5 s), small bladder responses were sometimes observed after short-lasting large responses during sneezing (Fig. 1A). Since similar pressure traces were also observed after opening the abdomen (Fig. 2A), these responses might be artifacts due to the movement of animals during sneezing. Thus we did not evaluate these long-lasting, small bladder responses for further data analyses. The response during individual sneeze episodes varied from 2.6 to 128.9 cmH2O depending on the strength of the sneeze (173 events in 5 rats). The average sneeze-induced bladder response was 36.6 ± 6.7 cmH2O (n = 5), and this reduced to 11.4 ± 0.7 cmH2O (n = 3) after opening of the abdomen (Figs. 2 and 3).
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Proximal urethra. The baseline reading of the microtip transducer
in the proximal urethra was not significantly different from the baseline in
bladder (Fig. 3A and
Table 1). The response in the
urethra increased during sneezing and returned to the baseline within 0.15 s
under an abdomen-closed condition (Figs.
1A and
3A). The time course
of the urethral response was similar to the time course of the bladder
response (Fig. 3A),
and the responses in the proximal urethra were lower compared with those in
the bladder (60% of the bladder response;
Table 1). The Ura-Ves
difference, which is the difference of urethral and bladder responses induced
by sneezing, showed mostly negative changes during sneezing, confirming that
the urethral response was generally lower than the bladder response (i.e., no
obvious positive change of urethral responses exceeding bladder responses;
Fig. 3A and
Table 1). After the abdomen was
opened, the urethral response during sneezing was significantly reduced to 3.0
± 0.6 cmH2O (n = 3; Figs.
2A and
3D). Middle
urethra. The baseline reading of the microtip transducer in the middle
urethra was significantly higher than the baseline in the bladder (Figs.
1B and
3B and
Table 1). During sneezing under
an abdomen-closed condition, the urethral response started 18.9 ± 5.5
ms before the bladder response (n = 5;
Fig. 3B) and reached a
peak value 14.7 ± 1.5 ms after the peak of bladder response (n
= 5; Fig. 3B).
Urethral responses measured by microtip transducer catheters during sneezing
were much longer in duration (10 times) than the bladder responses
(Fig. 3B), lasting
1.5 s. In addition, the urethral response during sneezing (44.5 ±
4.9 cmH2O, Table 1)
was 30% greater than the response in bladder. The average Ura-Ves
difference was positive during sneezing, showing that the sneeze-induced
urethral response was always higher than the bladder response (Figs.
1B and
3B and
Table 1). After the abdomen was
opened, the response measured by microtip transducer catheters in the urethra
still increased by 48.8 ± 8.1 cmH2O during sneezing
(n = 3; Figs.
2B and
3E), indicating that
the urethral response during sneezing was caused not only by passive
transmission of increased abdominal pressure but also by active reflex
contractions of the middle part of the urethra.
Distal urethra. The baseline reading of microtip transducer catheters in the distal urethra was higher than the baseline in bladder (Fig. 3C and Table 1). The reading in the urethra increased during sneezing and returned to the baseline in 0.15 s under an abdomen-closed condition, but the increase was significantly lower than the changes in the proximal or middle urethra (Figs. 1C and 3C and Table 1). The Ura-Ves difference was mostly negative during sneezing, and the maximum positive change was 3.0 cmH2O (Figs. 1C and 3C and Table 1). After the abdomen was opened, the urethral response measured by microtip transducer catheters during sneezing was 6.8 ± 1.1 cmH2O (n = 3; Figs. 2C and 3F).
Relationship between bladder and urethral responses. To examine
which part of the urethra is the most important for urinary continence, the
maximal readings of the microtip transducer catheters in the bladder and
urethra during sneezing under an abdomen-closed condition are plotted in
Fig. 4 (these data were from
the same experiments shown in Table
1). In the proximal and distal urethra, the maximal reading was
lower (60 and 50%, respectively) than the maximal reading in the bladder
during sneezing. In contrast, in the middle urethra the maximum reading was
higher (180%) than that in the bladder.
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We also examined whether active urethral closure responses in the middle
urethra can be affected by the strength of sneezing reflex
(Fig. 5). At least 10 sneeze
events were induced in each rat, and 64 sneeze events were measured in total
from five rats in the experiment shown in
Table 1. The increases in
Ura-Ves differences during sneezing were divided into eight groups according
to the size of bladder response from 0 to 80 cmH2O with each bin of
10 cmH2O. As shown in Fig.
5, no significant difference in the increase in Ura-Ves difference
was observed among groups. Even when the bladder response was <10
cmH2O, the increase in the Ura-Ves difference reached 40
cmH2O, indicating that the active urethral closure response during
sneezing was not affected by the strength of sneeze reflex.
Experiment 2: Effects of Bilateral Transection of Nerves Innervating Urethra on Urethral Responses During Sneezing
To investigate whether contractions of striated muscles such as the EUS and
pelvic floor muscles are responsible for the active closure responses in the
middle portion of the urethra during sneezing, the sneeze reflex was induced
under an abdomen-closed condition in rats whose pudendal nerves (n =
6), nerves to the iliococcygeous and pubococcygeous muscles (n = 7),
or both the pudendal nerves and the nerves to the iliococcygeous and
pubococcygeous muscles (n = 5) were cut bilaterally. The bladder
response during sneezing was not different among groups with or without nerve
transection (Table 2),
indicating that the degree of sneeze reflex was not different among groups.
However, the active component of the urethral closure response (i.e., increase
in Ura-Ves difference) during sneezing were significantly reduced by 67% after
bilateral transection of pudendal nerves
(Table 2). Although the
transection of the nerves to the iliococcygeous and pubococcygeous muscles
alone did not produce a significant change (34% reduction) in the urethral
active response, transection of both groups of nerves (pudendal nerves and
nerves to the iliococcygeous and pubococcygeous muscles) produced a further
significant reduction in the midurethral active response (additional 25%
reduction) compared with the pudendal nerve-transected group
(Table 2). After the two groups
of nerves (pudendal nerves and nerves to the iliococcygeous and pubococcygeous
muscles) were transected bilaterally, sneeze-induced responses in the middle
urethra were 35% of those in the bladder, with the time course of
response during sneezing being similar in the middle urethra and the bladder
(Fig. 6).
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The effects of bilateral transection of hypogastric nerves and visceral branches of pelvic nerves were also investigated (n = 3). The transection of these nerves did not affect the middle urethral response and Ura-Ves difference during sneezing (Table 2), indicating that these nerves were not involved in active urethral closure responses in the middle urethra during sneezing.
Experiment 3: Effects of Bilateral Transection of Pudendal Nerves on EMG Activity of EUS During Sneezing
To confirm the participation of the EUS in the sneeze reflex, EMG recordings of the EUS were compared before and after bilateral transection of the pudendal nerves under an abdomen-opened condition. EUSEMG activity was increased during sneezing (Fig. 7) and not altered in sham-operated animals (116 ± 23% of controls, n = 4). However, EUS-EMG activity was markedly decreased to 18 ± 9% of control values after bilateral transection of pudendal nerves (n = 3; P < 0.05, Student's t-test) compared with the sham-operated group. In addition, anal sphincter EMG activity was increased during sneezing (n = 3; data not shown), indicating that the sneeze-induced reflex activity is present not only in the EUS but also in the anal sphincter.
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Experiment 4: Effects on Sneeze Leak Point Pressure of Bilateral Transection of Pudendal Nerves and Nerves to Iliococcygeous and Pubococcygeous Muscles
To evaluate total urethral resistance generated against urinary leakage, leak point pressure during sneezing was measured using pressure transducers that were connected to bladder catheters inserted into the bladder from the dome. To clarify the contribution of nerve-mediated active urethral closure mechanisms to the total urethral resistance under a stress condition, sneeze leak point pressure was compared in the sham-operated group and the nerve-transected group (i.e., both pudendal nerves and nerves to the iliococcygeous and pubococcygeous muscles were transected).
Maximal intravesical pressure during each sneeze event varied from 11.3 to 40.8 cmH2O (sham-operated group; 220 events in 4 rats) and 8.2 to 40.0 cmH2O (nerve-transected group; 217 events in 4 rats), and the average value did not differ between the sham-operated and nerve-transected groups (20.5 ± 1.2 and 20.6 ± 1.0 cmH2O, respectively, n = 4 each). In all four sham-operated rats tested, no fluid leakage from the urethral orifice was observed during sneezing, which produced an increase in intravesical pressure reaching to as high as 37.0 ± 2.2 cmH2O. However, fluid leakage from the urethral orifice was observed in all four nerve-transected rats, with the incidence rate of leakage during sneezing being 53.7 ± 6.8%. Sneeze leak point pressures in nerve-transected rats averaged 16.3 ± 2.1 cmH2O.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The present study revealed that the urethral closure response during the sneeze reflex differed in proximal, middle, and distal portions of the urethra. In the proximal or distal urethra, the time course of the urethral response during sneezing was similar to the time course observed in the bladder, and responses in the proximal urethra did not exceed those in the bladder (i.e., no obvious positive increase in Ura-Ves difference) during sneezing. In addition, sneeze-induced urethral and bladder responses were markedly reduced when the abdomen was opened to eliminate the rise of abdominal pressure during sneezing. Taken together, these results suggest that a part of increased abdominal pressure is passively transmitted to the proximal or distal urethra without reflex urethral contractions.
However, the behavior of the middle urethra during sneezing was markedly different from the proximal or distal urethra. The present study revealed that 1) the duration of sneeze-induced response in the middle urethra was significantly longer than the duration of bladder response, 2) the Ura-Ves difference was high during sneezing, and 3) the sneeze-induced urethral response was still obvious after opening of the abdomen. These results indicate that the middle urethral response is mediated, at least in part, by active urethral closure mechanisms induced by sneezing. This is in line with the previous findings in dogs that sneezing induced a pressure increase in the urethra that was higher than pressures in the rectum or bladder and did not disappear after opening the abdomen (30, 31). In the dog, pressure increases in the urethra during sneezing disappeared when the urethra was shielded from the reflex contraction of pelvic floor muscles, indicating that active contractions of pelvic floor muscles during sneezing caused pressure increases in the urethra and must be important for maintaining urinary continence in dogs (10).
The present study also provided insights into the mechanisms underlying active urethral contraction during sneezing by examining the effects of somatic nerve transection on sneeze-induced urethral responses. It has been reported that the somatomotor branch of pelvic nerve (i.e., nerves to the iliococcygeous and pubococcygeous muscles) innervates pelvic floor muscles such as illiococcygeus muscles and pubococcygeous muscles in rats and that the pudendal nerve innervates the EUS and other pelvic floor muscles such as coccygeous muscles (16, 18). It has also been demonstrated that electrical stimulation of the pelvic nerve, which contains the nerves to the iliococcygeous and pubococcygeous muscles, increases EMG activity of iliococcygeus and pubococcygeous muscles (18), that electrical stimulation of pubococcygeous muscles increases intraurethral pressure (19), and that electrical stimulation of the pudendal nerve induces urethral contraction (14). On the basis of these findings, we examined the effects of bilateral transection of the pudendal nerves, the nerves to the iliococcygeous and pubococcygeous muscles, or both sets of nerves (pudendal nerves and nerves to the iliococcygeous and pubococcygeous muscles) and found that transection of pudendal nerves significantly reduced the sneeze-induced urethral responses measured by microtip transducer catheters as well as the Ura-Ves difference in the middle urethra during sneezing under an abdomen-closed condition. Moreover, the effects of combined transection of pudendal nerves and nerves to iliococcygeous and pubococcygeous muscles further reduced significantly the midurethral responses during sneezing compared with pudendal nerve-transected rats, while transection of iliococcygeous and pubococcygeous muscle nerves alone did not significantly reduce the sneeze-induced midurethral response. Therefore, these results suggest that somatic pathways in the pudendal nerves and the nerves to the iliococcygeous and pubococcygeous muscles are both significantly involved in the sneeze-induced active closure response in the middle urethra and that the contribution of pudendal nerve-mediated striated muscle activity to the continence mechanisms during stress may be greater than that of iliococcygeous and pubococcygeous muscle activity. This conclusion is further supported by the reduction in EMG activity of EUS muscles during sneezing after transection of bilateral pudendal nerves.
We also observed that, after transection of both groups of somatic nerves, the sneeze-induced response in the middle urethra was 65% lower than the bladder response, indicating that passive transmission of the sneeze-induced increased abdominal pressure to the middle urethra was less effective than the transmission to the bladder. This difference was also noted in the proximal or distal urethra.
As shown in Fig. 4, during a stress condition induced by sneezing, the urethral response exceeded the bladder responses only at the middle portion of the urethra, which exhibited both active and passive components of urethral closure mechanisms, but not in the other parts (proximal and distal) of the urethra, which showed only a passive pressure transmission mechanism. Thus it seems reasonable to assume that the middle urethra is most important for increasing urethral resistance under sneeze-induced stress conditions in the female rat.
However, because microtip transducers only measure the local force/unit area (mechanical stress) exerted by the tissue inner surface on the transducer tip, recorded values do not necessarily reflect the true urethral pressure (9, 21). Therefore, to examine the role of the active urethral closure mechanism mediated by activation of somatic nerves innervating the EUS and pelvic floor muscles in urinary continence, we measured sneeze leak point pressure, which is defined as the minimal intravesical pressure during sneezing that can open up the urethra and induce fluid leakage from the urethral orifice without reflex bladder contractions. We found that in normal rats, no urinary leakage occurred during sneezing that increased intravesical pressure as high as 37 cmH2O. However, in all rats in which the active closure mechanism was abolished by transection of both the pudendal nerves and the nerves to the iliococcygeous and pubococcygeous muscles, fluid leakage from the urethral orifice during sneezing was observed at a considerably lower leak point pressure of 16.3 cmH2O. These results indicate that the sneeze-induced active urethral closure mechanism in the middle urethra detected by microtip transducer methods is indeed critical for preventing stress urinary incontinence in rats. In addition, microtip transducer measurements of bladder and urethral responses seem to be useful to examine the detailed mechanisms of active urethral closure mechanisms even though microtip transducers may not measure the urethral pressure as strictly defined. The major contribution of striated muscle contraction to sphincteric function in the middle urethra has also been reported in a previous in vitro muscle strip study in female rats (2). It was demonstrated that 1) the circular muscle strip preparation of rat female urethra responds to electrical field stimulation in a succinylcholine-sensitive manner, which is indicative of striated muscle contractions, and 2) the most prominent muscular component in the middle urethra is circularly oriented striated muscle cells, while smooth muscle fibers dominate near the bladder orifice. In addition, it should be noted that the present study was not designed to investigate the bladder-to-urethral reflex, another important continence mechanism, because the experiments were performed in the emptied bladder condition (experiments 1 and 2) or in the pelvic nerve-transected condition (experiment 4). Thus further studies are needed to investigate the bladder-to-urethral reflex that contributes urinary continence.
Previous clinical findings also suggested the existence of active urethral closure mechanisms during stress conditions in humans. It has been demonstrated in healthy women that urethral closure forces during coughing were significantly reduced by bilateral pudendal nerve blockade (26). It has also been reported in humans that urethral pressure started rising before the pressure rise in the bladder during coughing and that this preceding pressure increment disappeared in women with SUI (27, 28, 33). The present study in rats also showed that during the sneeze reflex the response in the middle urethra preceded the bladder response. Similar arrangements of smooth and striated muscles have been described in female rat and human urethra (11); i.e, an inner longitudinal smooth muscle layer as a continuation of the bladder musculature, proximal circular smooth muscle layer, and circular or obliquely oriented striated muscle fibers starting from the midurethra distally. Thus it is assumed that the active urethral closure mechanism under stress conditions such as coughing and sneezing is important for preventing SUI in humans as well as animals.
Furthermore, it has been documented in women with SUI that 1) the number of pelvic floor muscle fibers that exhibit pathological damage increased (8), 2) fast-twitch type II muscle fibers in the pelvic floor decreased (4), 3) pelvic floor musculature showed evidence of partial denervation due to pudendal neuropathy (23, 24), 4) EMG activity of the striated urethral sphincter muscle decreased (25), and 5) the ability to voluntarily increase urethral pressure decreased (3). Thus it is possible that the active urethral closure mechanisms under stress conditions might be impaired in these SUI women, thereby leading to SUI. Recent studies have revealed that duloxetine, which is a 5-hydroxytryptamine and norepinephrine reuptake inhibitor, also increases EMG activity of EUS in the cat (12, 29) and is effective for treatment of SUI (17). Therefore, it is also possible that duloxetine might act by enhancing active urethral closure mechanisms, although further studies are necessary to clarify this point. Thus, as demonstrated in the present experiments, animal models that can explore the active urethral closure mechanism during stress conditions such as sneezing can be useful for studying the pathophysiology of SUI as well as developing new treatment modalities of SUI.
In summary, this study demonstrated that under stress conditions such as sneezing the rat urethra has active urethral closure mechanisms mediated by somatic nerve-induced reflex contractions of EUS and pelvic floor muscles, in addition to passive transmission of increased abdominal pressure, and that this active urethral pressure response during sneeze serves as an important mechanism for preventing SUI. Evaluation of these active urethral closure mechanisms using microtip transducer catheters and sneeze leak point pressure measurements could be useful techniques for developing new treatments of SUI.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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