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American Journal of Physiology - Renal Physiology logoLink to American Journal of Physiology - Renal Physiology
. 2019 Jul 17;317(4):F781–F788. doi: 10.1152/ajprenal.00197.2019

Analysis of continence reflexes by dynamic urethral pressure recordings in a rat stress urinary incontinence model induced by multiple simulated birth traumas

Joonbeom Kwon 1,2, Takahisa Suzuki 1, Ei-ichiro Takaoka 1, Nobutaka Shimizu 1, Takahiro Shimizu 1, Shun Takai 1, Satoru Yoshikawa 1, William C de Groat 3, Naoki Yoshimura 1,3,✉
PMCID: PMC6843045  PMID: 31313954

Abstract

The present study evaluated real-time changes in urethral pressure during the storage phase using a rat model with stress urinary incontinence (SUI) induced by simulated multiple birth traumas and investigated the relationship between urethral continence function and dynamic parameters associated with the changes in urethral pressure. Sprague-Dawley rats were divided into the following two groups: the sham group, which underwent three catheterizations of the vagina without distension at 2-wk intervals, and the vaginal distension (VD) group, which underwent three VDs at 2-wk intervals. After transection of the T8–T9 spinal cord, simultaneous bladder and urethral pressure recordings were performed during intravesical pressure elevation. Urodynamic parameters such as leak point pressure (LPP), urethral baseline pressure (UBP), maximum urethral pressure (MUP), the MUP-UBP differential (dUP) during intravesical pressure elevation, the bladder pressure when urethral contraction begins (Puc), and the bladder pressure at bladder neck opening (Pno) were then measured and compared. Compared with the sham group, LPP, UBP, dUP, MUP, Puc, and Pno were significantly decreased in the VD group. Pressure differences between LPP and Pno and between LPP and UBP (LPP-UBP) were also significantly different in the two groups. However, difference values of LPP and MUP or Pno and UBP were not altered after VD. Our new methods of simultaneous recordings of dynamic changes in bladder and urethral pressures are useful to fully evaluate the functional alterations in urethral continence function in the SUI model induced by multiple VDs. Moreover, LPP-UBP values, which correspond to the difference between Valsalva LPP and maximum urethral closure pressure in clinical urodynamics, would be useful to evaluate the impaired urethral continence function after simulated birth traumas in animal models.

Keywords: leak point pressure, maximum urethral closure pressure, rats, stress urinary incontinence, urethral pressure

INTRODUCTION

Stress urinary incontinence (SUI) is the pathological condition of involuntary urine leakage when abdominal pressure is increased. Currently, it is presumed that the urethral hypermobility caused by weakening of the structures supporting the urethra and/or reduced urethral pressure because of lowered urethral sphincter function work together in a complex manner (21, 24).

Urodynamic studies are not used for diagnostic purposes in all patients with SUI, but they are performed in some selected cases for excluding detrusor dysfunction, for establishing the postoperative prognosis, or for the purpose of research (21a). In clinical practice, Valsalva leak point pressure (VLPP) and abdominal LPP, which are, respectively, bladder pressure and abdominal pressure at urine leakage, have often been used to assess urethral function (17, 19); however, these are indirect measures of urethral function. Maximum urethral closure pressure (MUCP), which is used to measure urethral resistance directly, has been suggested as an indicator of intrinsic sphincter deficiency when below 20 cmH2O and as a possible preoperative predictor of surgical outcome (1, 7, 26).

However, MUCP alone cannot be used as a guideline for excluding or diagnosing SUI, and there is insufficient evidence to predict surgical outcome with MUCP alone (5, 10, 20). This may be because urethral pressure alters dynamically in response to the changes in bladder or abdominal pressure, and MUCP, which measures urethral pressure only under static conditions, does not reflect these dynamic changes. Hence, there have been some attempts to determine if urethral pressure measured separately at resting and stress conditions can predict urethral continence function (18). Moreover, in recent years, urethral pressure reflectometry has been tested to determine if the relationship between urethral pressure and abdominal pressure changes can estimate urethral continence dysfunction (14, 27). However, even using these clinically tested methods, it is not possible to measure real-time urethral pressure continuously. Therefore, there are no previous studies that have measured continuous real-time changes in urethral pressure under stress conditions. We therefore attempted to find a methodology using a SUI animal model that is clinically applicable for the assessment of continuous changes in bladder and urethral pressures. Using real-time bladder and urethral pressure recordings, we were particularly interested in determining the timing of bladder neck or urethral sphincter opening during intravesical pressure elevations that induce leakage. In our previous studies, a rat model of multiple birth traumas produced by three vaginal distensions (VDs) applied at 2-wk intervals was found to be useful for SUI research because it produced a durable decrease in LPP and urethral closing responses (28, 29, 31). In the present study, we used this SUI model to evaluate continuous changes in urethral pressure during bladder filling and to investigate the relationship between the urethral continence function and real-time changes in bladder and urethral pressures.

MATERIALS AND METHODS

Animals

Nulliparous female Sprague-Dawley rats (n = 34) weighing 220–280 g were used. All experimental protocols were conducted in accordance with National Institutes of Health guidelines and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Animals were divided into the following two groups: the sham group, which underwent three catheterizations of the vagina without distension at 2-wk intervals, and the VD group, which underwent three VDs at 2-wk intervals. Two weeks after the final VD or sham treatment, bladder and urethral pressures were measured simultaneously during a gradual increase in bladder pressure.

Vaginal Distension

Under anesthesia with pentobarbital (45 mg/kg ip), a 10-Fr balloon catheter (5 ml, Loganville, GA) with the tip cut off was inserted into the vagina, and the vaginal opening was sutured to prevent the catheter from slipping out of the vagina. The balloon catheter was inflated with a 4-ml saline infusion for a 1- to 2-min duration and then kept for 4 h to distend the vagina according to the methods in previous studies (28, 29, 31). In rats with three VDs that were performed at 2-wk intervals, we did not observe posttreatment signs of excessive pain or distress, including behavioral changes or weight loss.

Surgical Procedures

Just before the cystometric measurement, the spinal cord was transected at the level of T8–T9 under isoflurane anesthesia to block the spino-bulbo-spinal voiding reflex during bladder distension. In this condition, the bladder-to-urethral continence reflex organized in the lumbosacral spinal cord was preserved (11). After the ureters were exposed via a midline abdominal incision, both ureters were cut and ligated. Thereafter, a polyethylene catheter (PE-60, Clay Adams, Parsippany, NJ) was inserted into the bladder through the dome. After a small incision was made in the colon wall, feces were removed from the distal colon.

Measurement of Cystometric Parameters

After surgery, isoflurane anesthesia was replaced by urethane anesthesia (1.2 g/kg sc, Sigma, St. Louis, MO). Rats were placed in a supine position, and a saline reservoir mounted on a metered vertical pole was connected to the PE-60 catheter inserted into the bladder. LPP was measured using a modified intravesical pressure clamp method and defined as the bladder pressure at which fluid leakage occurred when intravesical pressure was gradually increased in 2.5-cmH2O steps from 0 cmH2O (4). A 3.5-Fr nylon microtransducer-tipped catheter (SPR-524, Millar Instruments, Houston, TX) was then inserted into the midurethra at 10−14 mm from the urethral orifice where maximal pressure changes in the urethra were obtained during changes of intravesical pressure (11). The microtransducer-tipped catheter was connected to a pressure transducer (Transbridge 4M, World Precision Instruments). Bladder and urethral pressures were acquired using Chart 5 software (ADInstruments, Colorado Springs, CO) on a PowerLab (ADInstruments) computer system equipped with an analog-to-digital converter. Thereafter, simultaneous measurements of bladder and urethral pressure were conducted for 1–2 h as terminal experiments followed by euthanasia. The various parameters of bladder and urethral pressure changes during passive intravesical pressure elevation were measured twice consecutively, and mean values were used as experimental data.

Experiments

Experiment 1: effects of urethral catheter insertion.

Sham and VD rats (n = 5 each) were used to validate the methodology and to confirm that LPP values were not affected by insertion of the urethral catheter to measure urethral pressure.

Experiment 2: comparison of urethral pressure responses in bladder neck-opened and -ligated conditions in sham and VD rats.

Sham and VD rats (n = 6 each) were used to evaluate urethral pressure responses in the bladder neck-ligated condition. At 2 wk after the last VD or sham operation, the bladder neck was ligated with a thread to isolate bladder and urethral pressure responses and thereby allow an evaluation of intrinsic urethral pressure profiles during intravesical pressure elevation without an effect of fluid outflow from the bladder to urethra. Also, another set of sham and VD rats (n = 6 each) was used to evaluate urethral pressure responses in the bladder neck-opened condition. Pressure parameters in these rats were compared with those in rats with bladder neck ligation.

Experiment 3: changes in bladder and urethral pressure responses after simulated birth traumas.

Bladder and urethral pressures were measured simultaneously with the bladder neck open in sham and VD rats (n = 6 each) at 2 wk after the last VD to determine which pressure parameters are affected by multiple VDs and whether VD alters urethral sphincter function.

Statistical Analysis

All the results are presented as the mean ± standard error and analyzed using IBM SPSS Statistics version 21 (IBM Corporation, Armonk, NY). Statistical analyses by unpaired t-test were performed to compare the parameters in sham and VD groups. For statistical analyses of the changes in measurements in the same rat within each group, a paired t-test was used. P value < 0.05 was considered significant for all analyses.

RESULTS

Effects of Urethral Catheter Insertion (Experiment 1)

In the sham group, mean values of LPP and LPP with a urethral catheter (LPPc), which were measured without and with the urethral catheter, were 39.5 ± 1.5 and 41.0 ± 2.3 cmH2O, respectively (Fig. 1A). In the VD group, mean values of LPP and LPPc were 29.5 ± 1.8 and 30.0 ± 1.6 cmH2O, respectively (Fig. 1B). There were no significant differences between these two measurement methods of LPP in the sham group (P = 0.426) or in the VD group (P = 0.778), indicating that urethral catheter insertion did not affect LPP values during passive intravesical pressure elevation. Thus, the following experiments were performed in the urethral catheter-inserted condition.

Fig. 1.

Fig. 1.

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Comparison between leak point pressure without the urethral catheter (LPP) and with the urethral catheter (LPPc) in the same rats. P = 0.426 in the sham group (A) and P = 0.778 in the vaginal distension (VD) group (B) as determined by a paired t-test.

Evaluation of Bladder and Urethral Pressure Responses in Sham Rats

Figures 2 and 3 show representative bladder and urethral pressure traces under bladder neck-opened and -ligated conditions, respectively, during passive intravesical pressure elevation in sham rats. In the bladder neck-opened condition (Fig. 2), the following parameters were measured: LPPc (Fig. 2, point A), urethral baseline pressure (UBP; Fig. 2, point B), bladder pressure at which urethral contractions start (Puc; Fig. 2, point D), and bladder pressure at which the bladder neck begins to open (Pno; Fig. 2, point E). UBP was measured after urethral pressure remained stable for more than 2 min at a bladder pressure of 0 cmH2O. With the bladder empty, average UBP was 23 cmH2O and remained stable as bladder pressure was increased to above 20 cmH2O, after which (Fig. 2, point D) it gradually increased during graded increases in bladder pressure until fluid leakage occurred from the urethral orifice (Fig. 2, point A). In the bladder neck-ligated condition (Fig. 3), urethral pressure also remained stable as bladder pressure was increased to ~20 cmH2O, after which (Fig. 3, point D) it started to increase and rose abruptly to a plateau (Fig. 3, point F) that was maintained despite the continued increase in bladder pressure. The abrupt increase in urethral pressure evoked by bladder pressures in excess of 20 cmH2O between points D and F in Fig. 2 was attributable to a bladder-to-urethral reflex because passage of fluid between the bladder and urethra is blocked by the ligature. These results also indicate that in the bladder neck-opened condition, the bladder neck opens beyond point F in Fig. 2, allowing fluid to leak into the proximal urethra as the bladder fills, producing the continuous rise in urethral pressure, which is absent in the bladder neck-closed condition.

Fig. 2.

Fig. 2.

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Representative recordings of simultaneous bladder and urethral pressure measurements in a sham animal during passive intravesical pressure elevation in the bladder neck-opened condition. B is an expanded portion of A, as indicated by the rectangular box with an increased timescale. The following parameters were evaluated: leak point pressure with a urethral catheter (point A), urethral baseline pressure (point B), differential values of urethral pressure during intravesical pressure elevation (point C), bladder pressure when urethral contraction begins (point D), bladder pressure when the bladder neck begins to open (point E), the point at which the bladder neck is opened (point F), and maximum urethral pressure (point G). Because urethral pressure continues to elevate beyond point F in the bladder neck-opened condition, it is considered that the bladder neck opens at point F to induce continued urethral pressure elevation with urethral pressure fluctuations because of fluid leakage into the proximal urethra.

Fig. 3.

Fig. 3.

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Representative recordings of simultaneous bladder and urethral pressure measurements in the bladder neck-ligated condition in a sham rat. B is an expanded portion of A, as indicated by the rectangular box with an increased timescale. Urethral pressure began to increase when bladder pressure exceeded baseline urethral pressure (point D). Thereafter, urethral pressure was gradually increased as the bladder further distended, and urethral pressure then plateaued from point F and did not further increase any more, even though bladder pressure continued to increase. Beyond point F, synchronous bladder and urethral pressure fluctuations were observed (B); however, the amplitudes of pressure fluctuations were smaller compared with those in the bladder neck-opened condition (Fig. 2B).

In the bladder neck-ligated condition, synchronous small pressure fluctuations in the bladder and urethra could be observed after point F in Fig. 3, suggesting that bladder distension and/or intrinsic bladder contractions induce reflex activity to elicit these urethral contractions. In the bladder neck-opened condition, bladder and urethral pressure fluctuations also occurred but with larger amplitudes (Fig. 2B) compared with those in the bladder neck-ligated condition (Fig. 3B), suggesting that bladder and urethral reflex activity is exaggerated by fluid flow through the bladder neck to the urethra. Thus, the beginning point of urethral pressure fluctuations presumably coincides with bladder neck opening (Fig. 2, point F) and correlates with the point (Fig. 3, point F) at which urethral contraction pressure reaches a plateau in the bladder neck-ligated condition. Therefore, we defined the intravesical pressure value just before the urethral pressure fluctuations start as Pno (i.e., bladder pressure at which the bladder neck begins to open; Fig. 2, point E) in the bladder neck-opened condition and measured maximum urethral pressure (MUP; Fig. 2, point G) just before urethral pressure fluctuations began. Additionally, the pressure difference between MUP and UBP, which is termed as the differential values of urethral pressures during intravesical pressure elevation (dUP; Fig. 2, point C), was evaluated.

Comparison of MUP and dUP in Bladder Neck-Opened and -Ligated Conditions in Sham and VD Rats (Experiment 2)

In the sham group, the mean value of MUP was 32.5 ± 1.4 and 28.8 ± 1.4 cmH2O (P = 0.141) in bladder neck-opened and -ligated conditions, respectively. The mean value of dUP was 9.3 ± 0.7 and 8.4 ± 0.5 cmH2O (P = 0.470) in the bladder neck-opened and -ligated conditions, respectively. Therefore, there were no differences in MUP or dUP values between the bladder neck-opened and -ligated conditions in the sham group (Fig. 4A). The results were similar in the VD group, in which there were no significant differences between mean values of MUP in bladder neck-opened (18.5 ± 0.6 cmH2O) and bladder neck-ligated conditions (16.9 ± 1.3 cmH2O, P = 0.284) or mean values of dUP in bladder neck-opened (3.3 ± 0.4 cmH2O) and bladder neck-ligated conditions (3.2 ± 0.4 cmH2O, P = 0.882; Fig. 4B). These results indicate that the parameters related to the urethral continence function such as MUP and dUP are also reliable even when they are measured in the bladder neck-opened condition.

Fig. 4.

Fig. 4.

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Comparison of urethral pressure parameters between two experimental groups with the opened and ligated bladder neck in the sham (A) and vaginal distension (VD) groups (B). Statistical analyses were performed by an unpaired t-test. dUP, differential values of urethral pressures during intravesical pressure elevation; MUP, maximum urethral pressure.

Changes in Bladder and Urethral Pressure Responses After Simulated Birth Traumas (Experiment 3)

The detailed results shown in Table 1 indicate that LPPc, UBP, dUP, Puc, Pno, and MUP were significantly decreased in the VD group compared with the sham group. The differences between LPPc and Pno (LPPc-Pno) and between LPPc and UBP (LPPc-UBP) were also significantly lower in the VD group than in the sham group. However, the differences between LPPc and MUP (LPPc-MUP) and between Pno and UBP (Pno-UBP) were not significantly changed by VD.

Table 1.

Comparison of parameters between sham and VD groups in the bladder neck-opened condition

Parameters Sham Group VD Group P Value
LPPc, cmH2O 42.5 ± 1.8 28.3 ± 1.4 0.001
UBP, cmH2O 23.1 ± 1.1 15.3 ± 0.5 0.001
dUP, cmH2O 9.2 ± 0.9 3.3 ± 0.4 0.001
Puc, cmH2O 21.5 ± 1.3 16.7 ± 1.1 0.019
Pno, cmH2O 28.0 ± 0.9 19.2 ± 1.2 0.001
MUP, cmH2O 32.3 ± 1.7 18.5 ± 0.6 0.001
LPPc-MUP, cmH2O 10.2 ± 1.5 9.8 ± 1.3 0.830
Pno-UBP, cmH2O 4.9 ± 1.0* 4.0 ± 0.9* 0.522
LPPc-Pno, cmH2O 14.5 ± 1.5 9.2 ± 1.4 0.027
LPPc-UBP, cmH2O 19.4 ± 1.4 13.1 ± 1.5 0.012
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All values are presented as means ± SE; n = 6 animals in the sham group and 6 animals in the vaginal distension (VD) group. Statistical analysis was performed by an unpaired t-test. LPPc, leak point pressure with a urethral catheter; UBP, urethral baseline pressure; dUP, differential values of urethral pressures during intravesical pressure elevation; Puc, bladder pressure at which urethral contractions start; Pno, bladder pressure at which the bladder neck begins to open; MUP, maximum urethral pressure; LPPc-MUP, the pressure difference between LPPc and MUP; Pno-UBP, the pressure differences between Pno and UBP; LPPc-Pno, the pressure difference between LPPc and Pno; LPPc-UBP, the pressure difference between LPPc and UBP.

*

Pno-UBP values were set to 0 cmH2O for statistical analysis in one sham rat and one VD rat, which showed negative values in this parameter.

DISCUSSION

In a previous study (12), it was found that two reflexes are involved in the urethral continence mechanism. One reflex is the bladder-to-urethral continence reflex, in which bladder afferent pathways activated by intravesical pressure elevation induces the urethral closure response (11), and the other reflex is the reflex that is activated by neural pathways in the central nervous system during stress conditions, such as sneezing, to directly activate the urethral closure response (13).

In the present study, we designed the experiments to focus on bladder-to-urethral reflex mechanisms under conditions in which bladder pressure is easily and reliably controlled (4). To evaluate the bladder-to-urethral continence reflex during the storage phase, the T8–T9 spinal cord was transected before functional measurements to block the voiding reflex triggered by the signals passing between the brain stem and spinal cord, which hinders the continuous observation of urethral continence activity during bladder distension. Under this condition, the urethral continence reflex in the spinal cord activated by bladder afferent pathways during bladder filling is still preserved because the nerves that constitute bladder-to-urethral reflexes, such as the pelvic nerves, pudendal nerves, hypogastric nerves, and nerves to pelvic floor muscles, remain intact (11).

Several animal models have been introduced for studying the pathophysiology of SUI, using VD (6, 31), ovariectomy (15), urethrolysis (25), electrocauterization of the urethra (3), and pudendal nerve transection (9). The urethrolysis, nerve transection, or electrocauterization-induced SUI models are suitable for studying the pathophysiology of intrinsic sphincter deficiency elicited by denervation, the ovariectomy model is useful for studying the estrogen deficiency-related mechanism of SUI, and the VD model has an advantage in studying the birth trauma-related SUI pathophysiology because birth trauma injuries can induce damages of both pelvic floor muscle and external urethral sphincter functions because of pudendal nerve injury. However, the SUI condition in the single VD model is usually short lasting (28, 29). In a recent study (31), we performed VDs three times at 2-wk intervals and found that urethral dysfunction was longer lasting, suggesting that the multiple VD model is more appropriate as a SUI model compared with the single VD model. In the present study, we confirmed that the multiple VD group compared with the sham group exhibited decreased urodynamic parameters related to urethral pressure responses such as UBP, dUP, and MUP as well as LPP. In addition, there are some limitations in our rat model of SUI. First, rats are quadruped animals whose bladder and urethra are supported by a lower anterior abdominal wall, whereas in a biped like humans, they are cradled by pelvic floor muscles, including levator ani muscle. Therefore, it is likely that the transmission of abdominal pressures to the urethral region during stress conditions is different between these two species. To address this problem in posture differences between rodents and humans, a previous study (4) using rats examined urethral parameters such as LPP in a vertical position, whereas the present study was performed in a supine position to minimize the movement of intraurethral microtransducer-tipped catheters. Nevertheless, because this study examined bladder-to-urethral reflex activity during bladder distension rather than passive pressure transmission during abdominal pressure elevation, the results of our study in rats could still be applicable to the urethral continence reflex mechanisms in humans. Second, the estrous cycle status was not taken into account in this study. Because the estrogen level greatly contributes to urethral continence function as shown in animal models of estrogen deficiency induced by ovariectomy (15), further studies are needed to evaluate the effects of estrogen levels altered during the estrous cycle on urethral continence reflexes.

The major aim of the present study was to record the real-time changes of urethral pressure during passive intravesical pressure elevation and to investigate which urodynamic parameters are most related to urethral continence function. For this purpose, bladder and urethral pressures were measured simultaneously with catheters inserted into the bladder and urethra. Because this novel method of simultaneous recordings might affect urodynamic parameter values compared with those obtained by the conventional bladder catheter-only method, we first compared the values in two different conditions with or without urethral catheter insertion (experiment 1). In experiment 1, LPP and LPPc were measured consecutively before and after catheter insertion in the same rat to examine if urethral catheterization affects values in LPP measurements. As shown in the results (Fig. 1), urethral catheter insertion for simultaneous urethral pressure recordings did not affect LPP values in either sham or VD groups. However, in some clinical studies, it has been reported that urethral catheter size affects maximum flow rate (Qmax) and detrusor pressure (Pdet), like PdetQmax, during urodynamic studies (16, 22). The possible reason for these different results could be that LPP in this study was measured by the passive leakage of urine but not by the active urine stream, whereas Qmax and PdetQmax are measured under the active voiding. Therefore, it is considered that urethral resistance was affected less by urethral diameter in this study.

To evaluate LPP values during simultaneous bladder and urethral pressure recordings, bladder filling should be performed without bladder neck ligation, unlike in previous methods (11). However, it is not known whether fluid leakage through the opened bladder neck affects the accuracy of microtransducer-tipped catheter measurements of urethral pressure. Therefore, experiment 2 was designed to address this question. Figure 2 shows simultaneous measurements of bladder and urethra pressures in the bladder neck-opened condition; Fig. 3 shows measurements of bladder and urethral pressures in the bladder neck-ligated condition. During the experiment with bladder neck ligation (Fig. 3), there was a significant disadvantage that LPP cannot be measured even though bladder pressure continuously increased, because no leakage through the urethra was observed. During the experiment under the bladder neck-opened condition (Fig. 2), it seems that the bladder neck opened (Fig. 2, point F) after urethral pressure reached MUP (Fig. 2, point G), which corresponds to the plateau level of increased urethral pressure during bladder distension in the bladder neck-ligated condition (Fig. 3). Also, at this point, synchronous fluctuations in bladder and urethral pressure recordings have larger amplitudes in the bladder neck-opened condition (Fig. 2, points E and F, respectively) compared with those in the bladder neck-closed condition (Fig. 3, point F). These results suggest that bladder and urethral reflex contractions are induced beyond this point (point F) and are enhanced because of fluid flow through the bladder neck to the urethra, although it is not known whether the increase in fluctuation pressure amplitudes in the bladder neck-opened condition is due to passive pressure transmission from the bladder to the urethra or urethral reflex activity induced by fluid inflow into the urethra. Comparison of pressure parameters such as MUP and dUP in bladder neck-ligated and -opened conditions showed that these parameter values were not different under the two different conditions. Therefore, these two parameters related to changes in urethral pressure seem to be useful to assess the urethral activity during bladder filling, even when experiments are conducted without ligation of the bladder neck.

As shown in Fig. 3, when bladder pressure exceeded the UBP, urethral pressure increased rapidly, which is thought to be due to contraction of the urethral sphincter muscles induced by the bladder-to-urethral reflex, which contributes to the maintenance of urinary continence. Because urethral pressure did not rise beyond MUP, although bladder pressure continued to rise in the bladder neck-closed condition, whereas after Pno in bladder neck-opened animals, urethral pressure steadily increased above MUP as bladder pressure was elevated until fluid leakage occurred, it is likely that the urethral opening process has two stages: initial bladder neck opening at MUP and subsequent opening of the urethral sphincter later at LPP.

The pressure measurements in the midurethra reflect the contractions of both urethral smooth muscles and external sphincter striated muscles (2, 8, 11, 30). However, the contribution of external sphincter muscles to UBP seems to be weak when the bladder is empty, because the transection of pudendal nerve did not reduce the UBP significantly (11). In addition, this two-stage process of urethral opening is in line with a recent study (8) using morphological analyses of the rat urethra, which showed that bladder neck opening occurred first followed by relaxation of urethral sphincter muscles during intravesical pressure elevation. Therefore, we evaluated the difference between bladder and urethral pressures for opening the bladder neck by measuring Pno-UBP, which showed a mean value of ~4.5 cm H2O, without significant differences between sham and VD groups (Table 1). These results suggest that smooth muscle activity, which closes the bladder neck during bladder filling, is relatively well maintained after multiple VDs.

If it can be assumed that proximal urethral pressure and bladder pressure are the same after the bladder neck opens, then our results indicate that even after bladder neck opening, leakage beyond the external urethral sphincter does not occur immediately. Leakage through the urethral orifice occurred when bladder pressure exceeded MUP to open the urethral outlet (Table 1). The pressure difference at this point was defined as LPPc-MUP in this study and was found to be ~10 cmH2O without significant differences between sham and VD groups (Table 1). However, LPPc-Pno was significantly lower in the VD group than in the sham group (Table 1). LPPc-Pno represents the change in bladder pressure from the timing of bladder neck opening to fluid leakage into the urethral opening (Fig. 2), which is an indirect indicator of external sphincter function. Thus, the lower LPPc-Pno value suggests that an impairment of external sphincter function exists after multiple VDs. This conclusion is supported by the significant reduction in the VD group of dUP, which is a direct indicator of external sphincter function (Table 1).

When considering the clinical translation of this study, it is speculated that UBP, MUP, and LPPc used in this study could be correlated with the following parameters used in clinical practice: UBP with MUCP under rest conditions, MUP with MUCP with a stress condition like abdominal straining or bladder filling, and LPPc with VLPP.

As shown in the present study, dUP, which is calculated by MUP-UBP (Fig. 2) and corresponds to the clinical parameter of MUCP at stress − MUCP at rest, could be used as a novel parameter predicting urethral continence function. Because it represents dynamic pressure changes but not static values such as MUCP or VLPP, dUP could be a more potent predictor for urethral continence function, although in clinical practice, measurement of MUCP at stress conditions has not been standardized, and reliable data have so far not been obtained. Furthermore, it was assumed that LPPc-UBP can also reflect urethral continence function, as verified in experiment 3 (Table 1). Given that LPPc and UBP used in this study can correspond to clinical parameters such as VLPP and MUCP at rest, respectively, VLPP-MUCP at rest can also be used to assess urethral continence function, particularly external sphincter function, although further clinical research is needed to validate this assumption.

In conclusion, the relatively long-lasting SUI condition produced by multiple VDs in rats is a reliable model to study the mechanisms underlying the lower urinary tract dysfunction caused by multiple birth-related injuries. The use of our new method of simultaneous recordings of bladder and urethral pressures to evaluate real-time changes in urethral continence function has revealed that external sphincter function reflected by changes in LPPc-UBP values, which correspond to the VLPP-MUCP value in clinical urodynamics, is impaired in a rat model of SUI with multiple simulated birth traumas.

ABBREVIATIONS

ALPP

Abdominal leak point pressure

dUP

Differential values of urethral pressures during intravesical pressure elevation

EUS

External urethral sphincter

ISD

Intrinsic sphincter deficiency

LPP

Leak point pressure without a urethral catheter

LPPc

Leak point pressure with a urethral catheter

MUCP

Maximum urethral closure pressure

MUP

Maximum urethral pressure

Pno

Bladder pressure at which the bladder neck begins to open

Puc

Bladder pressure at which urethral contractions start

SUI

Stress urinary incontinence

UBP

Urethral baseline pressure

VD

Vaginal distension

VLPP

Valsalva leak point pressure

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-107450.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.K., S.Y., and N.Y. conceived and designed research; J.K., T. Suzuki, E.T., and T. Shimizu performed experiments; J.K., T. Suzuki, E.T., N.S., and S.T. analyzed data; J.K., W.C.d.G., and N.Y. interpreted results of experiments; J.K. and T. Suzuki prepared figures; J.K. drafted manuscript; J.K., W.C.d.G., and N.Y. edited and revised manuscript; J.K., T. Suzuki, E.T., N.S., T. Shimizu, S.T., S.Y., W.C.d.G., and N.Y. approved final version of manuscript.

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