Stress urinary incontinence (SUI) is the common type of urinary incontinence with a significantly negative impact on quality of life in women.  Although the etiology of SUI in women seems to be multifactorial, multiple vaginal parities and estrogen deficiency are considered to be important risk factors of SUI in elderly women.  Thus, animal models of simulated birth trauma induced by multiple vaginal distention (VD) and estrogen deficiency induced by bilateral ovariectomy (OVX) have been used to study the SUI pathophysiology in rats.  It is well known that simulated birth trauma injury induced by single VD elicits a SUI condition in rats, which is, however, usually restored within 2 weeks.  In previous studies, multiple birth trauma injuries in rats significantly impairs urethral function, resulting in longer-lasting SUI [Ref.1].  It has also been reported in rats that OVX significantly impairs urethral function from the early stage (3 weeks), but does not induce SUI until the late phase (6 weeks). 

In rats, bilateral OVX reportedly induces morphological changes in the urethral epithelium [Ref.2].  It has also been reported that serotonergic (5HT) paraneurons uniquely located in the urethra can modulate urethral sensory function and that their numbers decrease in aged or diabetes rats [Ref.3].  However, the difference of changes in the urethra in these two models has not been well characterized, especially regarding epithelial morphology and the expression of 5HT+ paraneurons in the urethra.  We therefore evaluated the urethral histological changes of the epithelium and 5HT+ cell expression, and urethral continence function in rats with OVX-induced estrogen deficiency or VD-induced simulated birth trauma.

A total of 60 adult virgin Sprague-Dawley rats (8-12 weeks old) were used.  Rats were divided into; (1) sham operation group (laparotomy and wound closure) (N=12); (2) OVX 3-weeks group (N=12); (3) OVX 6-weeks group (N=12); (4) one-time VD group (N=12) [VD-1] ;(5) three-times VDs group (N=12) [VD-3].  VD was induced by balloon catheter inflation in the vagina (4ml, 4 hour) as previously described [Ref.1].  In the VD-1 group, VD was performed once 2 weeks before the evaluation. In the VD-3 group, VD was repeated every 2 weeks for 3 times, and the urethral evaluation was performed at 2 weeks after the final VD.  In the OVX group, the urethral evaluation was performed after 3 or 6 weeks of bilateral OVX.  

The surgical techniques for urethral function testing were same as previously described [Ref.1].  The bladder was exposed through an abdominal incision, ureters were cut bilaterally, and their distal ends were ligated.  The visceral branches of the pelvic nerves were transected bilaterally near the internal iliac vessels to prevent reflex bladder contractions.  Urethral function was evaluated by a 3.5-Fr nylon catheter with a side-mounted microtransducer located 1 mm from the catheter tip in 6 rats of each group.  The catheter was inserted into the middle urethra (10 to 15 mm from the urethral orifice), at which the highest urethral baseline pressure (UBP) was obtained.  The microtransducer-tipped catheter was connected to a pressure transducer, and urethral responses were recorded with data-acquisition software on a computer system equipped with an analog-to-digital converter.  The catheter position was monitored throughout the experiments to confirm that the location of the transducer had not been changed.  After UBP became stable, approximately 20 min after the catheter insertion, sneezes were induced by an insertion of a rat’s whisker into the nostril; and the changes of urethral responses during sneezing were examined by measuring amplitudes of the urethral responses during sneezing (A-URS), which were determined as the maximal pressure change from the baseline in centimeters per H2O during sneezing.  

Following the urethral function analysis, the mid urethra was harvested, and the tissue sections (10 μm) were stained with Hematoxylin-eosin (H&E) and anti-5HT antibodies (rabbit anti-5-HT, 1:100, No. 20080, Immunostar, Houston, TX, and Alexa Fluor 488 Donkey Anti-Rabbit IgG, 1:400, ab150073, abcam, Cambridge, MA) for immunofluorescent detection of 5HT+ paraneurons.  Quantitative morphometric analysis was done by the image J software.

In addition, in separate groups of rats (n=6, each group), changes in urethral mRNA levels of tryptophan hydroxylase-1 (TPH1), a key enzyme of 5HT synthesis, were quantified and normalized by a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase ; GAPDH) using RT-PCR.  

All data are represented as means ±  SE.  Statistical significance was evaluated in each group vs. sham group using one-way ANOVA followed by Dunnett's multiple comparison test.  Thereafter, repeated ANOVA measures followed by Tukey's multiple comparison test for the comparison between two groups.  All data were analyzed using the JMP software (ver. 11; SAS Institute, Cary, NC).  P < 0.05 was considered significant.

In the 3-weeks or 6-weeks OVX groups, UBP was significantly decreased only in 6-weeks rats compared to sham rats (45.3% reduction, P < 0.05, Fig.1A).  Also, A-URS were significantly reduced in OVX 3-weeks (19.0% reduction) and 6-weeks (49.7% reduction) compared to sham rats (P < 0.05, Fig.1A).  In the single or multiple VD groups, UBP was significantly decreased only in VD-3 rats compared to sham rats (51.2% reduction, P < 0.001).  Also, A-URS was significantly decreased only in VD-3 rats compared to sham rats (51.1% reduction, P < 0.001, Fig.1A).  In histological analyses, the mean thickness of the epithelium of the mid-urethra was significantly decreased in OVX 3-weeks (30.2±2.18 µm), OVX 6-weeks (29.3±1.98 µm), VD-1 (54.1±2.21 µm) and VD-3 (29.9±2.07 µm) rats compared to sham rats (72.3±2.19 µm, P < 0.0001, Fig1.B,C).  The reduction of epithelial thickness in VD-1 rats were significantly milder compared to OVX 3, 6-weeks and VD- 3 groups (Fig.1C).  The number of 5HT+ paraneurons in the urethra wall was significantly decreased in OVX 3-weeks (10.1±0.64/mm2), OVX 6-weeks (9.48±0.60/mm2), VD-1 (13.6±0.93/mm2) and VD-3 (9.10±0.72/mm2) rats compared to sham rats (17.7±0.59/mm2, P < 0.001, Fig2.A,B).  The reduction of 5HT+ paraneurons in VD-1 rats was significantly milder compared to OVX 3, 6-weeks and VD-3 groups (Fig.2B).  In RT-PCR, relative mRNA levels of TPH-1 were significantly decreased in OVX 3-weeks (0.497±0.06), OVX 6-weeks (0.456±0.05) and VD-3 rats (0.469±0.06) compared to sham rats (1.000 ±0.15, Fig2.C, P < 0.01).  VD-1 rats exhibited a tendency in a decrease of TPH-1 expression (0.824±0.06), without statistical significance compared to sham rats (P = 0.598).

The two SUI models exhibited similar urethral dysfunction evident as reductions in UBP and urethral responses during sneezing.  The assessment of urethral structural changes in these two models also revealed that urethral epithelial atrophy and loss of 5HT+ paraneurons occur prior to urethral dysfunction inducing SUI in both models and that these changes occur rapidly at a greater degree from the early phase (3 weeks) of OVX, compared to VD groups, which showed the slower onset of these histological changes.  Thus, the tissue-damaging process in the urethra, which contributes to the establishment of SUI, could be different between VD-induced simulated birth trauma and OVX-induced estrogen deficiency.

Both multiple birth trauma and estrogen deficiency play significant roles in inducing SUI; however, the pathohistological process of these two conditions might be different.  Therefore, the combination of these two risk factors could accelerate the disease process of SUI in women.  In addition, the two SUI models used in this study would be suitable for the study of different aspects of the pathophysiological mechanisms of SUI.

Yoshikawa S, et al. Effects of multiple simulated birth traumas on urethral continence function in rats. Am J Physiol Renal Physiol (2017) 313:1089-1096.
dos Santos AR, et al. Morphometric analysis of the urethra of castrated female rats treated with tamoxifen. Maturitas (2008) 59:275-80
Cao N, et al. Streptozotocin-induced diabetes causes upregulation of serotonin (5-HT)2A/C receptors in lumbosacral cord motoneurons and down regulation of serotonergic paraneurons in the urethra. Brain Res (2019) 1715:21-26.