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Keywords:

  • bladder compliance;
  • PBOO;
  • rabbit;
  • MMP-1;
  • TIMP-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

What's known on the subject? and What does the study add?

  • A decrease in bladder compliance is known to be correlated with deterioration of renal function after bladder outlet obstruction (BOO). Increased deposition of extracellular matrix (ECM) in the detrusor layer is the primary reason for decreased compliance. In the bladder, as in other organs, ECM deposition is dependent on the balanced activity of proteolytic enzymes, including matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). The imbalance between MMPs and TIMPs is a key regulator in ECM turnover. It has been shown that an altered proteolytic balance between MMP-1 and TIMP-1 favours accumulation of ECM and decreases tissue compliance in an ovine fetal BOO model. Also, MMP-1 was significantly down-regulated, while TIMP-1 levels were increased, in a time- and pressure-dependent manner in a smooth muscle cell (SMC) mechanical strain model.
  • In the present study we measured the bladder compliance of control, sham-operated and partial BOO (PBOO) rabbits using a UDS-600 urodynamic testing machine. Collagen deposition between and within the detrusor SM bundles was evaluated using Masson's Trichrome stain and transmission electron microscopy. The expression levels of MMP-1 and TIMP-1 were evaluated by Western blot. We found that the imbalance between MMP-1 and TIMP-1 favours accumulation of extracellular collagen, the structural components associated with decreased bladder compliance after PBOO.

Objective

  • To investigate the underlying mechanisms of bladder compliance after partial bladder outlet obstruction (PBOO) and the role of the collagen degradation enzyme matrix metalloproteinase-1 (MMP-1) and its endogenous inhibitor tissue inhibitor of metalloproteinase-1 (TIMP-1) during this process.

Materials and Methods

  • Bladder compliance of control, sham-operated and PBOO rabbits was measured using a UDS-600 urodynamic testing machine.
  • Collagen deposition between and within the detrusor smooth muscle bundles was evaluated using Masson's Trichrome stain and transmission electron microscopy.
  • The expression levels of MMP-1 and TIMP-1 were evaluated by Western blot.

Results

  • Bladder compliance decreased after PBOO.
  • Collagen deposition increased both between and within the detrusor smooth muscle bundles, and had an inverse relationship with bladder compliance.
  • MMP-1 and TIMP-1 expression negatively correlated with bladder compliance.

Conclusion

  • These findings indicate that the imbalance between MMP-1 and TIMP-1 favours accumulation of extracellular collagen, the structural components associated with decreased bladder compliance after PBOO.

Abbreviations
ECM

extracellular matrix

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

MMP

matrix metalloproteinase

PBOO

partial BOO

SM(C)

smooth muscle (cell)

TIMP

tissue inhibitor of metalloproteinase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

In older men BPH is a common disease, with >50% of men aged ≥50 years experiencing some degree of BOO secondary to BPH [1, 2]. To understand the effect of BOO in humans, several animal models have been developed using different species [3-6]. The rabbit bladder provides an excellent model to investigate the physiological, histological, and biochemical properties of a functioning bladder [6-9]. Similarities between partial BOO in animals and obstructive dysfunction in humans include increased bladder mass, reduced contractile ability and decreased compliance [10, 11].

A decrease in bladder compliance is known to be correlated with deterioration of renal function [12-14]. Increased deposition of extracellular matrix (ECM) in the detrusor layer is the primary reason for decreased compliance [15-17]. In the bladder, as in other organs, ECM deposition is dependent on the balanced activity of proteolytic enzymes, including matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) [18]. The imbalance between MMPs and TIMPs is a key regulator in ECM turnover.

The latent form of MMP-1 (interstitial collagenase) has been shown to be responsible for the degradation of the principal collagens of the bladder, namely collagen types I and III [19]. TIMP-1 is a 30 kDa glycoprotein that binds MMPs tightly with a 1:1 stoichiometry and inhibits all collagenase, gelatinase and stromelysin activities [20]. Peters et al. [18] showed that an altered proteolytic balance between MMP-1 and TIMP-1 favoured accumulation of ECM and decreased tissue compliance in an ovine fetal BOO model. In addition, Backhaus et al. [21] showed that MMP-1 was significantly down-regulated, while TIMP-1 levels were increased, in a time- and pressure-dependent manner in smooth muscle cell (SMC) mechanical strain model. However, the relationship between bladder compliance and the expression of MMP-1 and TIMP-1 in a partial BOO (PBOO) rabbit model remains unknown.

In the present study we examined bladder compliance, the volume fraction of collagen and correlated them with MMP-1 and TIMP-1 expressions in rabbit detrusor SM bundles after long-term PBOO.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

The protocol for this study was approved by the Institutional Animal Care and Use Committee of Xi'an JiaoTong University. Male New Zealand White rabbits (4-month-old) were separated into three groups: normal (n = 10), sham-operated (n = 10), and PBOO (n = 20). The urinary bladder outlet was partially obstructed as described. Briefly, under anaesthesia, an 8 F catheter was inserted into the bladder via the urethra. Then, the bladder neck was exposed through a small vertical abdominal incision. Once the ureters were identified, a 2-0 silk suture was placed below the bladder neck with a right angle clamp. To maximise standardisation of the PBOO, a second 8 F catheter was placed outside the urethra, and the silk suture was tied around both catheters, which were then removed. In the sham-operated group the silk suture was cut and removed after an identical dissection.

Urodynamic Studies

At 4 weeks after surgery, control, PBOO and sham-operated rabbits were anesthetised and the obstructive ligature was removed. The bladder was catheterised with modified 6 F double-lumen urodynamic catheters. The bladder was emptied of all residual urine and then the intravesical pressure was monitored using a UDS-600 urodynamic testing machine (Laborie Medical Technologies, Mississauga, ON, USA). A slow-fill cystometrogram was performed at 4.9 mL/min until overflow incontinence occurred. The bladder compliance was defined as ΔV/ΔP. After two PBOO rabbits died of unexplained diarrhoea, a total of 18 PBOO, 10 control and 10 sham-operated rabbits underwent urodynamic testing.

After the urodynamics study, the rabbits were humanely killed and the bladder was harvested via the same midline incision, during which a 1 × 1.5 cm section of the ventral bladder wall was excised with a scalpel. Three full thickness strips were fixed for histology and ultrastructure studies. The mucosal and serosal layers were removed, and the SM layer was isolated and flash frozen in liquid nitrogen for protein extraction.

Histology

Full-thickness sections of bladder tissue from each rabbit were immediately fixed in 10% neutral-buffered formalin for 12 h and embedded in paraffin blocks. Sections were cut (5 μm thick) from each block and were used for staining for SM and collagen using Masson's Trichrome. The blue stained collagen and red counterstained muscle was then highlighted for each image. The percentage of tissue stained for each colour was calculated and data were acquired from the stained cross-sections using Image Pro Plus 5.0 image analysis software. The percentage area was selected in the program and generated automatically for each image. In all, 20 random images per tissue section were analysed for each tissue sample. The averages were then calculated for each group.

Electron Microscopy

Each bladder strip was immersed in 4% glutaraldehyde in 0.1 m phosphate buffer and, following fixation, trimmed into 1–2 mm cubes and post-fixed in 1% aqueous osmium tetroxide for 2 h. Specimens were then dehydrated and embedded in Epon-812 resin. Semi-thin (1-μm thick) sections from each block were stained with toluidine blue and examined by light microscopy to select the most appropriate blocks for thin sectioning. Thin sections were cut, mounted on uncoated copper grids, double stained with uranyl acetate, followed by lead citrate, and examined in a Hitachi-600 transmission electron microscope.

Protein Extraction and Western Blotting

Total protein was extracted in extraction buffer (20% glycerol, 50 mm Tris-HCl [pH 6.8] and 0.5% [v/v] Tween 20) supplemented with a protease inhibitor cocktail (Sigma). After adding 10% SDS, the sample was mixed, boiled for 4 min, and centrifuged at 10 000 rpm (9391RCF) for 15 min to remove undissolved material. The protein concentration of the supernatant was measured using the Bradford method. Aliquots of protein extract containing 30 μg of total protein were electrophoresed in a 10% SDS-PAGE gel. After transferring onto a nitrocellulose membrane (Osmonics), the membrane was cut and blocked for 1 h with PBS containing 5% dry milk and 0.1% Tween 20, and then incubated with primary antibodies against MMP-1 (Santa Cruz, catalog no. sc-6837, 1:500) and TIMP-1 (Santa Cruz, catalog no. sc-6832, 1:200) overnight at 4 °C, followed by a secondary antibody (mouse anti-goat IgG at 1:5000). After extensive washing, the membrane was developed using ECL Plus (Amersham) and exposing the membranes to autoradiographic films (Kodak X-OMAT). The membrane was then stripped using stripping buffer (60 mm Tris-HCL pH 6.8, 0.7% β-mercaptoethanol, 2% SDS) and washing extensively, incubate with primary antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz, catalog no. sc-20358, 1:3000) and follow the protocol above to check the GAPDH expression. Films were scanned and analysed with Image J Software (The National Institutes of Health).

Statistical Analysis

All the data are expressed as the mean (sem) and P < 0.05 was considered to indicate statistical significance. anova followed by a Bonferroni post hoc test for individual differences was used for comparative purposes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

Bladder Compliance

There was no difference between the compliance of control and sham-operated rabbit bladders, which was defined as normal. Normal rabbit bladder compliance was between 4–6 mL/cmH2O. Compared with the control and sham-operated rabbits, the compliance of the PBOO bladders was lower.

Based on the compliance levels, the PBOO bladders could be divided into three groups: (i) bladder compliance levels of 2–3.5 mL/cmH2O; (ii) bladder compliance levels of 1–2 mL/cmH2O; and (iii) bladder compliance levels of <1 mL/cmH2O (Fig. 1A). Because the protocol for BOO and the urodynamic study were the same, we assumed the differences in the compliance levels in the PBOO group were due to the individual differences. Figure 1B shows the representative cystometry figures of normal and group 3 PBOO bladders. Figure 1B I shows the normal bladder compliance of sham-operated rabbits, in which the intravesical pressure increased very slowly, while Fig. 1B II shows the compliance levels of PBOO bladders, in which the intravesical pressure increased slowly at the beginning due to filling of the corresponding volume of residual urine, then increased very sharply, indicating significantly decreased bladder compliance.

figure

Figure 1. Bladder compliance values for control, sham and PBOO rabbits. (A) Each point represents one bladder compliance value. Based on the obtained bladder compliance values, rabbits in the PBOO bladder group could be subdivided into three groups: group 1(G1), bladder compliance levels of 2–3.5 mL/cmH2O; group 2(G2), bladder compliance levels of 1–2 mL/cmH2O; group 3(G3), bladder compliance levels of <1 mL/cmH2O. (B) Representative cystometric figures for sham and low compliance bladders.

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Histology

Figure 2 shows representative images of control, sham-operated and PBOO rabbit bladder tissue sections with Masson trichrome stained SM and collagen. In the control and sham-operated rabbit bladders, the space between the SM bundles was wide, and there was little collagen deposition between the SM bundles. In the PBOO group, collagen deposition was greater between the SM bundles.

figure

Figure 2. Distribution of SM and collagen in the anterior wall of control, sham and PBOO rabbit bladders. (A) control and (B), sham rabbit bladders, showing small amounts of collagen. (C) group 1; (D), group 2; and (E), group 3 PBOO bladders showing increased amounts of collagen. Magnifications are indicated by the bars in the figures. (F) quantitative data showing the relative area of collagen for each condition. Each bar represents the mean ± sem of the group. *<0.05.

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In group 1 PBOO bladders (Fig. 2C), collagen deposition was slightly augmented between the SM bundles, but the SMs still appeared normal. In group 2 PBOO bladders (Fig. 2D), the space between the SM bundles was wider and collagen deposition was significantly greater. In group 3 PBOO bladders (Fig. 2E), the SM seemed to break down and collagen deposition was significantly greater. The whole bladder of group 3 PBOO rabbits appeared as a fibrous silkworm cocoon, with a thin bladder wall and decreased bladder weight (data not shown). The effect of PBOO on the relative volume of collagen is shown in Fig. 2F. Bladder compliance was decreased from group 1 to group 3 PBOO bladders, and we observed more collagen deposition from group 1 to group 3 PBOO bladders, suggesting that this rabbit model was good to study bladder compliance.

Ultrastructure

We then studied collagen deposition within the SM bundles. Figure 3 shows representative ultrastructural images of control, sham-operated and PBOO rabbit bladder body detrusor tissue. The detrusor SMCs in the control and sham-operated bladders had a fine structure, with little collagen infiltration between the SMCs. Intercellular spaces were somewhat wider than the controls in group 1 PBOO bladders and they were occupied by clusters of collagen fibrils. In group 2 and group 3 PBOO bladders, the spaces between adjacent SMCs were enlarged and the deposition of collagen was significantly greater (Fig. 3D,E).

figure

Figure 3. Representative ultrastructure images. (A) control and (B), sham SMC outlines are regular and fine. (C) In group 1 PBOO bladders, the SM appeared normal and the interspace between the SMCs increased slightly. (D) group 2 PBOO bladder SMCs were irregular and collagen was deposited in the wide intercellular spaces. (E) group 3 PBOO bladders showed an increasing amount of collagen deposition, and the SM was atrophied and broken down. Original magnification ×3000.

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Western Blot Analysis of MMP-1 and TIMP-1

Figure 4A shows the expression of MMP-1 and TIMP-1 in the control and PBOO rabbit bladders by Western blot. Semi-quantified data obtained from Western blot analysis are shown in Fig. 4B. The expression of MMP-1 diminished gradually in the PBOO groups. However, there was a significantly more expression of TIMP-1 (an important inhibitor of MMP activity) in the PBOO groups. The highest level of TIMP-1 expression was seen in group 3 PBOO bladders.

figure

Figure 4. Western blotting analysis showing the expression of MMP-1 and TIMP-1 in control, sham and PBOO rabbit bladders. (A) MMP-1 expression was reduced after PBOO, while TIMP-1 expression was greater.1, control; 2, sham; 3, group 1; 4, group 2; 5, group 3. (B) Semi-quantitative data based on Western blot results. Each bar is the mean ± sem of the group. *P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References

As in many disease states, the use of an appropriate animal model has greatly enhanced our understanding of the factors responsible for pathophysiology. Despite its acute onset, PBOO in the rabbit induces detrusor remodelling similar to that seen in men with BPH, in terms of its impact on structural and functional alterations in SM [22]. In the present study, we found that our rabbit model was an acceptable model to study bladder compliance after PBOO.

A previous study, investigating in situ whole bladder function using conscious rabbits, showed that maximum micturition pressure increased after PBOO [23]. We found that it was very difficult for us to perform these experiments in conscious rabbits because intravesical and intraperitoneal pressure changed irregularly when the rabbits struggled. Therefore, we performed the present urodynamic studies in anesthetised rabbits. As the purpose of the present study was to measure bladder compliance, we did not measure bladder contraction. Thus, the rabbits did not need to remain conscious. Under anaesthesia, intraperitoneal pressure was stable and did not significantly influence intravesical pressure, and therefore we did not measure intraperitoneal pressure. Due to the small bladder of the rabbit, we truncated a catheter normally used for a child's bladder, so the distance between the measure hole and the fill hole was only ≈1 cm.

We found that the bladder compliance of the control and sham-operated rabbit bladders was 4–6 mL/cmH2O, which is similar to values found in previous studies [24]. After 4 weeks of PBOO, bladder compliance was reduced by varying degrees, which is most likely due to individual differences between rabbits.

We found a close relationship between bladder compliance and collagen deposition [15-17]. Increased synthesis and deposition of connective tissue is an important characteristic of bladder dysfunction after PBOO. The present results showed that collagen deposition increased after PBOO. Samples from different groups of PBOO bladders had varying degrees of collagen deposition levels between the muscle fascicles, with bladder compliance reducing from group 1 to group 3 PBOO bladders. In contrast, the relative collagen area increased from group 1 to group 3 PBOO bladders. Taken together, these results indicate an inverse relationship between bladder compliance and collagen deposition. In addition, we examined collagen deposition within SM bundles and found that collagen infiltrated into the SM bundle, was deposited between the SMCs after PBOO, and had a close relationship with bladder compliance.

Excess ECM deposition is dependent on the balanced activity of proteolytic MMPs and their endogenous inhibitors TIMPs. MMPs constitute a family of zinc endoproteases that share structural domains and are capable of degrading ECM components [25]. MMPs are broadly classified into four different subgroups: (i) the collagenases (MMP-1, -8, and -13); (ii) the gelatinases (MMP-2 and -9); (iii) the stromelysins (MMP-3, -7, -10, and -11); and (iv) membrane-type MMPs (MT 1–4 MMP). Due to their potentially destructive nature, MMP activity is tightly regulated at different levels, including transcriptional control, secretion from the cell as inactive precursors and inhibition by the TIMP family, of which four members have been identified to date (TIMP-1, -2, -3, and -4) [26].

MMP-1 has been shown to breakdown collagen types I and III, the major ECM components of the bladder [19]. The present results showed that MMP-1 was significantly down-regulated after PBOO, while TIMP-1 levels increased, indicating a potential net ECM deposition in PBOO bladders. These alterations in MMP-1 and TIMP-1 favour ECM accumulation, which could contribute to the structural components associated with decreased bladder compliance.

In conclusion, the relative volume of collagen was increased after PBOO in rabbit bladders and was mainly distributed in the interfascicular planes. In bladders with severely reduced compliance, collagen infiltrated into the SM bundles and deposited between the SMCs. MMP-1 levels were down-regulated after PBOO, while TIMP-1 levels increased to a similar degree. These findings indicate that the imbalance between MMP-1 and TIMP-1 favours ECM accumulation, which leads to decreased bladder compliance after PBOO.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. References
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