Hypothesis / aims of study
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 have been reported to be important risk factors for 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 single simulated birth trauma injury induced by VD elicits SUI condition in rats, which is usually restored within 2 weeks. A previous study reported in rats that multiple birth trauma injuries significantly impairs urethral function, resulting in longer-lasting SUI [Ref.1]. We also reported in rats that OVX significantly impairs urethral function from the early stage (3 weeks), but does not induce SUI until the late stage (6 weeks) [Ref.2]. However, it has not been well characterized how VD and OVX differently alter histological and molecular profiles in the urethra. We therefore evaluated changes in the urethral function as well as structure and molecular expression in VD and OVX models by quantitative morphometric analysis and RT-PCR.
Study design, materials and methods
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]. The number of animals per group was 6 in urethral function and histology, and 6 in molecular studies. 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 [Ref.2]. Urethral function was evaluated by urethral baseline pressure (UBP), which was measured by a microtransducer-tipped catheter inserted into the mid-urethra (10 to 15 mm from the urethral orifice) from the external urethra.
The mid urethra was harvested at the end of the pressure analyses, and the tissue sections (10 μm) were stained with Hematoxylin-eosin (H&E) and Masson's trichrome (MT). MT-stained slides were analyzed quantitatively, in which the muscle layer was stained red, whereas the connective tissue was stained blue. The muscle/collagen ratio was determined in the whole urethral circumference in at least two non-contiguous sections (magnification x 40) per animal using the color segmentation tool in the image J software, which distinguishes regions stained with different colors and accurately measures the areas of muscle and collagen to yield a muscle/collagen ratio.
Also, changes in mRNA levels of urethral extracellular matrix (ECM)-related molecules such as collagen, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) 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 urethral functional analyses, the OVX 3 weeks and VD-1 groups did not show any significant change in UBP compared with the sham group. On the other hand, the OVX 6 weeks and VD-3 groups showed significant decreases in UBP values compared with the sham group (Fig.1A). In addition, the decrease in UBP of the VD-3 group was statistically significant compared with the VD-1 group (Fig.1A).
In histological evaluation, a layer of outer circular striated muscle fibers encircled the inner longitudinal smooth muscle layers (Fig.1B.). Striated and smooth muscle atrophy and disruption were moderate in the OVX 3 weeks group and severe in the OVX 6 weeks group. Morphometrical studies with Masson's trichrome staining also revealed the significantly decreased muscle ratio in OVX groups, due to the muscle atrophy (Fig.1C.), which was more evident in the OVX 6 weeks group than in the OVX 3 weeks group. VD groups produced muscle atrophy and collagen deposition, being more evident in the VD-3 group than in the VD-1 group. The proportion of collagen area was significantly increased in the VD-3 group compared with the sham or VD-1 group (Fig.1C).
mRNA levels of collagen 1a and collagen 3a were significantly increased in both VD-1 and VD-3 groups while MMP9 and TIMP1 were significantly increased only in the VD-3 group compared with the sham group (Fig.2).
On the other hand, mRNA levels of MMP2 were significantly decreased in the urethra in the OVX 3 weeks, OVX 6 weeks, VD-1, and VD-3 groups compared with the sham group. No significant changes in mRNA levels of TIMP2 were observed in OVX 3 weeks, OVX 6 weeks, VD-1 and VD-3 groups compared with the sham group (Fig.2).
Interpretation of results
In this study, OVX 6 weeks and VD-3 groups showed significant decreases in UBP values compared with the sham group, which were compatible to previous reports [Ref. 1,2]. In histological analyses, rats with estrogen deficiency induced by OVX exhibited morphological changes characterized by profound muscle atrophy in the urethra, whereas urethral collagen deposition was a major morphological characteristic in a rat model of multiple simulated birth injuries induced by VDs. These changes are likely to be involved in impairment of the urethral continence function in two different etiological conditions of SUI. Furthermore, these functional and morphological changes after VD or OVX were also associated with the urethral remodeling process in connective tissues evident as altered expression levels of ECM-related molecules including increased collagen expression, which is more apparent after multiple simulated birth injuries (i.e., VD-3 group). The upregulation of MMP9 has been considered to play a role in the breakdown of ECM molecules such as collagen. In this study, a high increase in mRNA of MMP9 was also observed in the VD-3 group. Previous studies reported that MMP9 was increased in the tissues such as denervated muscle and injured nerves [Ref.3]. The significant increase of MMP9 in association with upregulation of collagen transcripts in the urethra from VD-3 rats may suggest that the turnover of collagen synthesis and breakdown is accelerated after multiple VDs. The ECM remodeling due to MMP9 overexpression is involved in the impairment of the urethral continence function evident as decreases in UBP after multiple VDs.