Urinary frequency, urgency and urgency incontinence are common bothersome storage symptoms in men with partial bladder outlet obstruction (pBOO) due to BPH. Bladder function is altered following pBOO and often not completely be restored , even after the obstruction is relieved.  Although both sexes have been used for pBOO rat models, the female rat has been used more commonly because of the simpler anatomy and the absence of the accessory sex organs. In addition, most of the previous studies using male BOO rats evaluated only in early phase such as 2-6 weeks after pBOO production while functional, histological, and molecular expression changes in the chronic phase have not been well elucidated. Therefore, the purpose of this study was to investigate the progressive changes at different time points up to 12 months after pBOO using a male rat model, which is physiologically relevant with the pBOO condition in men with BPH.

A total of 68 adult male Sprague-Dawley rats (8-weeks old) were used. Rats were divided into: (1) sham operation group (N=20); (2) BOO 4 weeks group (N=20) [BOO 4w]; (3) BOO 12 weeks group (N=28) [BOO 12w]. BOO was produced under isoflurane anesthesia. After a lower abdominal incision, a 4-0 silk suture was placed around the urethra, including a metal rod with an outer diameter of 1.2mm placed extraluminally at the urethrovesical junction level proximal to the urethral fenestration [Ref.1].  After tying the suture, the rod was removed.  Sham operated animals were used as controls. At 4weeks (4w) or 12 weeks (12w) after inducing BOO, conscious rats were assessed by cystometrograms (CMG) with the urethral ligature intact. Saline solution was infused at 0.04–0.3 ml/min for approximately 1 h until rhythmic bladder contractions became stable. In this study, intercontraction intervals were adjusted to around 10-15 min intervals to evaluate the same number of voiding cycles because bladder capacity was variable following pBOO among animals.

The following CMG parameters were evaluated; bladder contraction amplitude (the peak pressure minus the basal pressure during each contraction period); baseline pressure (the pressure immediately after the reflex contraction); threshold pressure (the pressure immediately before the reflex contraction). Voided volume (VV) was determined by collecting voided saline. After the VV was determined, saline infusion was stopped and the residual volume (RV) was measured by withdrawing intravesical fluid through the catheter by gravity. The bladder capacity (BC) was calculated as VV + RV.  Voiding efficiency (VE) was calculated as (VV/BC) × 100. Bladder compliance was calculated as bladder capacity divided by the difference in threshold pressure and baseline pressure values in CMG, which were obtained after bladder emptying to eliminate the effect of PVR on bladder compliance. Non-voiding contractions (NVCs) were defined as bladder contractions with amplitudes >4 cmH20 prior to micturition.  Average values of each rat were calculated by the mean of two or three voiding cycles.  Some of the rats showed urinary retention during CMG, and these rats were excluded from the analysis of CMG parameters and histology.  Following cystometry, the rat bladder tissues were harvested and weighed. Bladder specimens were fixed in 4.0% buffered paraformaldehyde, embedded in OCT compound and  sectioned at 10 μm thickness. Bladder tissue sections were stained with Hematoxylin-eosin (H&E) and Masson's trichrome (MT). The muscle layer was stained red, whereas the connective tissue was stained blue in MT-staining. The collagen/muscle ratio was determined in the 10 random fields (magnification x100) from each bladder section 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 [Ref. 2].

Also, changes in mRNA levels of extracellular matrix (ECM)-related molecules such as collagen, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) in bladder mucosa and muscle layers were quantified with RT-PCR normalized by a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase ; GAPDH)

Totally, 14 of 20 sham rats, 14 of 20 BOO 4w rats and 22 of 28 BOO 12w rats underwent CMG. After exclusion of urinary retention rats, 14 sham rats, 10 BOO 4w rats and 8 BOO 12w rats were analyzed for CMG parameters and histology. The remaining 6 rats of each group were used for molecular studies. 
All data are represented as means ± SE. Statistical significance was evaluated among groups using one-way ANOVA followed by Dunnett's multiple comparison test. Thereafter, repeated ANOVA measures followed by Tukey's multiple comparison test were used 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.

The prevalence of urinary retention was 29 % (4/14) and 64 % (14/22) in 4w and 12w BOO rats (P = 0.0378), respectively (Fig.1A). Bladder weight of BOO rats with urinary retention was significantly greater compared to non-retention, moderate BOO rats or sham rats (Fig.1B).  

In CMG, contraction amplitudes and the number of NVCs were significantly increased in 4w BOO rats compared to 12w BOO rats or sham rats (Fig.1C, D). On the other hand, bladder compliance and capacity were significantly increased in 12w BOO rats compared to 4w BOO rats or sham rats (Fig.1D). Voiding efficiency (VE) was decreased in BOO rats vs. sham rats, being more evident in BOO 12w than in BOO 4w.

In the sham-operated rat bladder, collagen fiber staining pattern was predominant in the lamina propria, rather than in the interfascicular region. Morphometrical studies with MT staining revealed the significantly increased muscle ratio in BOO 4w, due to the muscle hypertrophy (Fig.2A, B). BOO 12w exhibits not only muscle hypertrophy, but also collagen deposition between muscle fibers (Fig.2A). The proportion of collagen area in BOO 12w was significantly increased compared with sham or BOO 4w (Fig.2B).

Overall difference of mRNA levels was more prominent in the blabber muscle layer than the mucosa (Fig.2C). Relative expressions of MMP2, collagen 1a (col1a), collagen 3a (col3a), and TIMP2 were significantly increased in the bladder muscle layer of BOO 4w rats compared with sham or BOO 12w rats (Fig.2C). On the other hand, mRNA levels of MMP9 and TIMP1 were significantly decreased in the muscle layer of BOO 12w rats compared with sham rats (Fig.3).

Structurally and functionally, pBOO causes a series of histological and biochemical changes in the bladder wall, through a process of initial inflammation, subsequent hypertrophy and ultimately fibrosis in the decompensation stage [Ref. 3].

The present results suggest that chronic pBOO at 12 weeks leads to a higher prevalence of urinary retention, larger bladder size, lower amplitudes of bladder contractions and fewer NVCs compared with 4 weeks pBOO. These data seem to be compatible to progression from the overactive to underactive phases in pBOO-induced bladder dysfunction [Ref. 3]. The histology confirms that this functional progression is associated with a pathological deposition of collagen in the detrusor muscle layer.  Also, upregulation of collagen1a, 3a and MMP2 in BOO 4w may be interpreted as a beginning phase of bladder remodeling and compensation process in pBOO.

The functional, histological and molecular comparison of early and chronic pBOO models of male rats is useful to understand the pathophysiology for the progression of bladder dysfunction related to BOO in men with BPH.

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