To use a rabbit model of partial bladder outlet obstruction (BOO) to investigate the point at which obstructive bladder dysfunction becomes irreversible.
To use a rabbit model of partial bladder outlet obstruction (BOO) to investigate the point at which obstructive bladder dysfunction becomes irreversible.
Partial BOO was induced in New Zealand White rabbits. It was then reversed and the rabbits were allowed to recover for 4, 8 or 12 weeks. Both at the time of reversal and at the end of the study, the rabbits were grouped according to bladder decompensation level (mild, intermediate or severe) based on bladder mass (weight).
A strong correlation was observed between the production and distribution of collagen and the reduction of smooth muscle contractile function. We found that only in the bladders that were severely decompensated at the time of reversal did collagen levels not decrease.
The data show that recovery of function after reversal of partial BOO is directly related primarily to collagen levels at the time of reversal.
The urinary bladder is made up of smooth muscle with a mucosal lining that protects the smooth muscle from the contents of the urine. Its function is to collect and store urine at a low intravesical pressure while periodically emptying via a coordinated contraction of the bladder body and relaxation of the bladder base and urethra . Bladder function depends on multiple factors, including the structure of the organ, its state of innervation, the contractile response of the smooth muscle to autonomic stimulation and the availability of metabolic energy. Bladder function is associated with each of these factors and a change in one factor could induce changes in the others [1-3].
Partial BOO in the rabbit has been shown to be a reliable model for bladder dysfunction secondary to BPH in men [4-7]. More than 80% of men > 50 years of age have some degree of BOO secondary to BPH and experience bladder problems as a result of their enlarged prostates [8, 9]. Although BPH is the most common cause of BOO, other causes include carcinoma, urethral strictures, masses and detrusor–sphincter dyssynergia [10, 11]. Drugs are available to treat BPH and to limit the negative effects it can have on the bladder but, in severe cases, men must undergo prostate reduction surgery , the aim of which is to remove the portion of the prostate surrounding the urethra (the transitional zone) which may be contributing to the obstruction. Unfortunately, not all bladders fully recover from their initially decompensated state . The more severely decompensated bladders have a smaller chance of returning to their optimum function, so it is sensible to analyse the underlying factors that mediate both the initial decompensation and the recovery of the bladder after the surgery.
Collagen is a type of connective tissue that has been associated with bladder dysfunction [14-16]. The concentration and distribution of collagen in the obstructed bladder can be a marker of bladder dysfunction [17, 18]. There appears to be an inverse relationship between collagen levels and both contractility and smooth muscle content [17, 19].
We believe there are three aetiologies of obstructive bladder dysfunction which are all related to the ischaemia/reperfusion mediated by the bladder hypertrophy that is directly related to partial BOO [20, 21]. These aetiologies are: 1) oxidative stress resulting in the generation of free radicals and oxidative damage; 2) calcium overload resulting from the inability to remove intracellular free calcium after a contraction in a partially obstructed bladder, causing the activation of calcium-activated degradative enzymes such as calpain and phospholipase A2 (PLA2), increased activity of which has been observed after partial BOO [22-27]; and 3) the shift from smooth muscle to collagen as smooth muscle damage increases [22, 23, 28]. Collagen levels can serve as a valuable marker to characterise the level of obstructive damage during partial BOO as well as after relief from BOO .
One of the secondary markers for obstructive damage is smooth muscle myosin, which is one of the major contractile proteins for the bladder and has been shown to change isoforms and content after partial BOO [30-33].
The primary objective of the present study was to determine the major characteristics of obstructed bladders that do not recover after reversal surgery, using a rabbit model of partial BOO. The hypothesis was that the concentration and distribution of collagen at the time of reversal is the main reason that bladder function does not recover after reversal of obstruction.
All studies were approved by the Institutional Animal Care and Use Committee of the Stratton VA Medical Center, Albany, NY, USA.
The New Zealand White rabbit is an excellent model for the study of bladder function and dysfunction. The rabbit bladder is of sufficient size to allow easy urodynamic evaluation and its response to in vitro field stimulation and pharmacological agents is consistent from rabbit to rabbit. In addition, rabbits can be housed in sufficient numbers to perform statistically significant chronic experiments. As discussed above, progressive changes in bladder morphology, biochemistry and function observed after partial BOO in these rabbits are very similar to those changes found in men secondary to BPH-induced obstructive uropathy. Mature male New Zealand White rabbits were used for all studies.
Each rabbit was sedated with ketamine/xylazine (25 mg/10 mg, i.m.) with surgical anaesthesia being maintained with isoflurane (1–3%). Under sterile conditions, the urinary bladder was catheterised with an 8-F Foley catheter and exposed through a mid-line incision. Partial BOO was initiated by placing a 00-silk ligature loosely around the catheterised urethra. The catheter was removed and the incision closed in layers with 3-0 sutures. Sham surgeries were performed similarly to the obstructions: each sham-operated rabbit underwent the same procedures (catheterization, anaesthesia and surgery) as the obstructed rabbits except that no ligature was placed around the urethra.
After 8 weeks of partial BOO, each rabbit was anaesthetised as stated above and the bladder exposed. The ligature was surgically removed and the bladder was catheterised through the urethra and emptied. Bladder mass was calculated as if the bladder was a solid cylinder or as a solid sphere, depending on the shape and measurements. Using calipers, we measured bladder length and radius. The calculations used were for the volume of a solid cylinder: volume = (pi) (R2) × length. The volume of a solid sphere was: (pi) (R3). Weight was calculated as volume × density. Previous studies have shown that control bladders had a density of ∼1.05 and that the obstructed bladder density was ∼1.15 measured by water displacement. After measurements, the incision was closed in layers with 3-0 silk. Control rabbits received sham surgery and bladder mass/weight measurements. The shape of control bladders after emptying was most like a solid sphere, whereas previously obstructed bladders were shaped most like a solid cylinder.
The obstruction surgery can cause some scarring and thus some obstruction may still remain after removing the ligature. However, we observed that the rabbits' urination frequency increased after reversal surgery. Rabbits with no obstructions urinated approximately 3–5 times daily, whereas obstructed rabbits urinated ≥10 times daily.
A total of 32 rabbits were separated into four groups of eight. Group 1 served as sham controls. Groups 2–4 underwent mild partial BOO; these rabbits were obstructed for 8 weeks and then each underwent a reversal operation. The rabbits in groups 2–4 were killed after 4, 8 and 12 weeks of recovery, respectively. Sham-operated rabbits were killed as follows: two at 4 weeks, three at 8 weeks, and three at 12 weeks after sham reversals. There were no significant differences among the sham rabbits in the variables measured for any of the studies and the sham rabbits were evaluated as one group. Three rabbits died during the obstruction period and were replaced.
Based on bladder weight at the time of reversals and at the end of the recovery periods, the rabbits were grouped according to severity of decompensation level: mild, intermediate or severe. The mean weight of control bladders was 2.5 g, mildly decompensated bladders weighed 5–12.5 g, intermediately decompensated bladders weighed 12.5–25 g and severely decompensated bladders weighed >25 g . The distribution of decompensation severity for the recovery duration groups at the end of the experiment was as follows: in the 4-week recovery group four bladders were mildly, two were intermediately and two were severely decompensated; in the 8-week recovery group four bladders were mildly, two were intermediately and two were severely decompensated; and in the 12-week recovery group two bladders were mildly, two were intermediately and four were severely decompensated. There were not enough bladders of each severity in each duration group for them to be analysed individually. Analysing the bladders for severity of decompensation as a group, independently of recovery duration, provided reasonable standard errors and statistics. The range of bladder weights for the severity categories was based on the functional responses after 8 weeks of obstruction in an earlier experiment that did not involve reversals [22, 35, 36]. For each rabbit in each recovery group (4, 8 and 12 weeks) it was therefore possible to determine the magnitude of recovery (shift in severity group).
Immediately after these studies were completed, the bladder was emptied, freed from fat and connective tissue, excised and weighed. Three isolated full-thickness strips of bladder body were taken for in vitro contractile studies. The remainder of the bladder was separated by blunt dissection into muscle and mucosal compartments, with each compartment being frozen in liquid nitrogen and stored at −80°C for biochemical evaluation.
Each isolated strip was placed in 15 mL of warmed oxygenated Tyrodes solution and allowed to equilibrate for 30 min at 37°C. A passive length–tension curve was generated and the contractile response to field stimulation at 32 Hz was used to determine the passive length that allowed maximum active tension generation. At this length, the responses to field stimulation at 2, 8 and 32 Hz, and responses to ATP (1 mM), carbachol (20μM) and KCl (120 mM) were determined. Each strip was washed three times at 15-min intervals with fresh warmed oxygenated buffer between pharmacological additions.
We have found that partial BOO does not alter the effective dose that results in 50% maximal contraction (ED50) for any form of stimulation. For each form of stimulation only the maximum tension was recorded. All data were recorded on a Grass model D polygraph (Grass Instruments, Warwick, RI, USA) and the data were digitised and analysed using the Grass Polyview A-D & conversion system (Grass Instrument Co, Warwick, RI, USA).
It should be noted that, in our experience, performing dose–response curves for all agents leads to fatigue because of the duration of each experiment and this would make the data unreliable, especially with the obstructed bladder strips.
Collagen levels were determined on muscle samples using the QuickZyme (Leiden, The Netherlands) Total Collagen Assay chemical kit. Tissue samples were hydrolysed at 100 mg/mL in 6 M HCl and incubated for 20 h in a thermoblock at 95°C. Threefold dilutions were carried out on all tissue samples using 4 M HCL. The standard curve was prepared from a stock of 1.2 mg/mL in 12 M HCl. The curve was created with dilutions in 4 M HCl and had values of 0.0672, 0.448, 0.224, 0.112, 0.056, 0.028, 0.014 mg/mL and a blank. The kit required the use of clear 96-well plates and a fluorescence plate reader set at 570 nm using a SpectraMax Plus by Molecular Devices (Sunnyvale, CA, USA).
Frozen bladder muscle wall was homogenised on ice in homogenisation buffer (Higgins IP buffer in 1 M Tris-HCl pH 7.5) containing 1% Halt protease inhibitor using a Brinkmann Polytron PT-3000 homogeniser (Westbury, NY, USA). Each sample was centrifuged at 24 000g for 30 min at 4°C in a Sorvall RC 28S (Newtown, CT, USA). Then, 1 mL of the supernatant was drawn off and stored in a −80°C freezer for protein analysis. These samples were analysed for protein using the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).
For the Western blot analysis, the membranes were blocked with 5% non-fat milk in 0.05% Tween 20 in TBS (TTBS), incubated and rocked for 30 min at 37°C. Membranes were washed with deionised water and then incubated with the addition of the primary antibody for 45 min at 37°C. The following primary antibodies were used: monoclonal anti-myosin (smooth) heavy chain (Clone hSM-V), produced in mouse, catalogue number M7786; monoclonal anti-myosin (light chains 70 and 20 kDa; Clone MY-21), produced in mouse, catalogue number M4401 (Sigma-Aldrich, St Louis, MO, USA).
After treatment, the membranes were washed with TTBS and incubated with the secondary antibody then washed again with TTBS. By washing five times for 5 min each at room temperature any unbound antibodies were removed. Each blot was then covered with ECL Plus substrate (Thermo Scientific) for 5 min, sealed in a plastic protector sheet, scanned and analysed with a Kodak Image Station 440CF and Kodak ID image analysis software.
The optical densities for both light and heavy chain myosin were normalised to control = 100% to better visualise the differences among the groups and to compare the groups statistically. Since partial BOO has been shown to alter the ratios of these proteins, we did not think that standard curves of the pure proteins would be of any value.
Biochemical assays for PLA2 and calpain were conducted on the muscle samples of the bladder . The samples were homogenised in a 50 mM Tris buffer (pH 7.8) at a concentration of 100 mg/mL. These samples were then analysed using an EnzChek Phospholipase A2 kit (Invitrogen, Carlsbad, CA, USA) and an SensoLyte 520 Calpain Activity Assay Kit (AnaSpec, Fremont, CA, USA). Both assays were conducted in clear 96-well plates using a fluorescence microplate reader. The excitation/emission for both PLA2 and calpain were read at wavelengths of 485–528 nm. For PLA2 and calpain, kinetics readings were taken every 2 min for 20 min and every 5 min for 1 h, respectively, to obtain the maximum velocity. Protein assays were conducted on all of the samples to normalise the data by μg of protein.
When analysed by duration of recovery there were eight rabbits per group. When analysed by decompensation severity (which included all obstructed rabbits) there were 10 mildly, six intermediately, and eight severely obstructed rabbits. All eight control rabbits were analysed as one group. Statistically, the recovery duration groups of data were analysed separately from the decompensation severity groups of data.
Statistical analysis of the following experimental datasets was performed individually: maximum contractile response to 1) field stimulation; 2) maximum contractile response to carbachol; 3) maximum contractile response to KCl; 4) maximum contractile response to ATP; 5) calpain activity; 6) PLA2 activity; 7) collagen concentration per mg/g tissue; 8) collagen concentration per mg bladder; 9) myosin light chain density for 70 kDa; 10) myosin light chain density for 40 kDa relative to control = 100%; 11) myosin heavy chain density relative to control = 100%; 12) malondialdehyde (MDA) concentration for muscle; and 13) MDA concentration for mucosa.
Each individual analysis used one- or two-way anova followed by the Tukey test for individual differences among datasets.
Figure 1 shows the bladder weights recorded at the time of reversal and at the end of the experiment, based on both the recovery period (duration of reversal) and the decompensation level (severity of dysfunction) at the time of reversal. It should be noted that in the case of severity of decompensation, the groups (mild, intermediate and severe) are composed of all three durations of reversals. Based on duration, the weights at reversal were all similar with very high standard errors because of the mix of mild, intermediate and severe obstruction among each of the duration groups. The bladder weights at the end of the experiment were significantly higher than the control bladder weights, and the weights at the end of the 12-week recovery period were significantly higher than those in all other groups.
Based on decompensation severity at the time of reversal, the bladder weights were progressively greater at the time of reversal. Interestingly, the weights at reversal and at the end of the experiment were similar for the mild and intermediate groups. The weights at the end of the experiment were all significantly higher than the control bladder weights. The weights of the severe decompensation group were significantly higher than all other severities.
The contractile response of the strips of bladder to field stimulation is shown in Figures 2A and B. In Figure 2, which was grouped by duration of recovery period, all three groups showed significantly less response than the control group. The largest contractile response of the strips was observed at 32 Hz. The ‘Obst’ group comprised rabbits with 8 weeks of obstruction without reversal taken from our previous study for comparison purposes only . Figure 2 shows the rabbits grouped by decompensation severity and shows that contractile response in all groups was significantly lower than that of the control. The responses of the mild and intermediate groups were roughly the same, while the response in the severe group was significantly lower than the control and the other two groups.
The contractile responses to carbachol, KCl, and ATP are shown in Figures 3A and B. The responses were very similar to that of the field stimulation, with each group showing a significantly lower contractile response compared with the control. Carbachol generated the largest response, followed by KCl and then ATP. Similarly, the severely decompensated group showed significantly lower responses for carbachol and KCl than all other groups.
In Figures 4A and B, calpain and PLA2 activities are presented by duration of recovery and severity, respectively. When comparing duration (Fig. 4A), the only group that showed a significant increase in calpain activity compared with the control was the 4-week recovery group. No group showed a significant difference in PLA2 activity. In Figure 4, calpain activity increased as decompensation level increased from mild to intermediate to severe, with only the severe decompensation group showing a significant increase compared with the control group. Similarly to the results shown in Figure 3, there were no significant differences in PLA2 activity among the groups.
The collagen concentrations are shown in Figures 5A and B. When analysing collagen concentrations (mg/g tissue) among the different recovery periods, there was a significantly lower collagen concentration in the 12-week recovery group. When analysed by decompensation severity, a significantly higher collagen concentration was observed in the severely decompensated bladders. In Figure 5, mg of collagen per bladder was analysed in the samples. A higher collagen concentration was found in all the recovery groups compared with the control group but the collagen concentrations in the recovery groups were not different from each other. If looked at by decompensation severity, only the intermediate and severe decompensation groups had significantly higher collagen concentrations compared with the control group, with the severely decompensated group showing a substantially higher concentration than that in all other groups.
Figures 6 and B show the optical density of myosin light chain among the different recovery duration groups and among the decompensation severity groups. The mild and intermediate decompensation groups had a significantly higher optical density for 70 kDa of myosin light chain while the intermediate and severe decompensation groups had significantly lower optical density with 20 kDa of myosin light chain than the control group. When comparing the different recovery periods, the 8-week and 12-week recovery groups had significantly higher optical density with 70 kDa of myosin light chain and all three recovery groups had significant lower optical density than the control group with 20 kDa of myosin light chain.
Figure 7 shows the optical density of myosin heavy chain according to decompensation severity and recovery period. The 8- and 12-week recovery groups had significantly higher optical density compared with the control and 4-week recovery groups. When analysed by severity, the mildly decompensated group had significantly higher optical density than control, and the 12-week recovery group had significantly lower optical density than the control group.
When grouped by duration of recovery, there were no differences among the groups with regard to the bladder weight at the time of partial BOO reversal; however, each bar has an extremely large standard error indicating that for each recovery period the severity of dysfunction was very variable. We expected the bladder weight at the end of the experiment to show a progressive decrease as the recovery period increased; however, this was not the case, as the bladder weight in the 12-week group was significantly higher than in any of the other groups.
As expected, when all rabbits were separated by severity according to bladder weight at the time of partial BOO reversal, there was a progressive increase in both the weight at reversal and the weight of the bladder at the end of the experiment, especially in the severely decompensated group. It is interesting to note that the bladder weight of the severely decompensated group decreased significantly and substantially from the weight at the time of reversal.
The contractile response to field stimulation grouped by duration and severity showed that the dysfunctional condition of the bladder, characterised by its inability to contract as effectively, was more closely related to severity than duration. This has been shown in previous studies . It might be assumed that bladders that were given 12 weeks to recover would regain more of their contractile function, but this was not the case in the present study, as recovery at all durations was equal. This experiment suggests that the decompensation state of the bladder dictates the contractile impairment and that the recovery time was less important because each recovery group contained samples of different severities of bladder decompensation. Another explanation could be that the bladder undergoes most of its recovery before the 4-week period and therefore no differences would be seen among the 4-, 8- or 12-week recovery groups. The first explanation seems more likely, because the severe decompensation group showed the least amount of response to all forms of stimulation as these bladders were the most severely decompensated at the time of reversal. Interestingly, the rabbits with mildly and intermediately decompensated bladders recovered to approximately the same degree, whereas the rabbits with severely decompensated bladders recovered to a significantly lesser degree.
The increased levels of calpain seen in the severely decompensated bladders shows that calpain (calcium dysregulation) may play an important role in severe decompensation of the bladder. In a previous study, the level of calpain decreased during the recovery process when compared with that after 8 weeks of obstruction . The 4-week group still showed a high level of calpain as a result of the ischaemia and reperfusion induced by the obstruction. The levels then decreased with longer recovery times and were not significantly different from the control. This suggests that calpain is not preventing obstructed bladders from recovering fully after a reversal. PLA2 showed no significant differences when plotted in a graph by either severity or duration, suggesting that it plays no major role in bladder recovery.
When analysed by levels of decompensation, the amount of light chain myosin in the rabbit bladders shows that the 20 kDa light chain myosin had a significantly lower optical density in the intermediately and severely decompensated bladders. This shows that as a bladder becomes more severely decompensated, the amount of light chain myosin decreases and will affect the rate and force of contraction within the bladder smooth muscle. When analysing light chain myosin by recovery time, the data show that the 70 kDa light chain myosin was able to recover with an increasing recovery time, although no recovery was shown for the 20 kDa chain. The 4-, 8- and 12-week groups all showed a significant decrease in optical density when compared with the controls. This supports the theory that the 20 kDa light chain myosin has more of an effect on the decompensation and recovery of the bladder.
For heavy chain myosin, data show that with a longer period of recovery there was a significant increase in the optical density for the 8- and 12-week recovery period. With regard to the severity groups, there was a significant increase in optical density in the mildly decompensated group compared with control which reduced below control in the severely decompensated group. This information leads us to believe that heavy chain myosin may not play as important a role in decompensation as light chain myosin and may not affect the bladder until it reaches a high rate of decompensation.
Analysing the amount of collagen per g of tissue by severity showed that collagen levels were significantly and substantially increased in the severely decompensated bladders but not in the mildly or intermediately decompensated bladders. This correlates very well with the greater bladder weight at reversal and at the end of the experiment for this group. It also most likely plays a major role in the decreased contractile function in this group. Thus, the marked increase in collagen in the severely decompensated bladders calculated either by mg/g tissue or per bladder is a major factor preventing the bladder from recovering from the decompensated state. Looking at the distribution of collagen histologically using trichrome stain clearly shows the connective tissue is between individual smooth muscle cells as well as between bundles, which severely prevents the bladder from contracting . These data provide strong support for our original hypothesis.
To summarise the research experiment, the obstruction and reversals of bladders have shown that some bladders regain function while others will remain in a state of severe decompensation. In most cases, obstructed then reversed bladders will never regain full function and will instead remain at some level of decompensation. It was shown that collagen levels did increase while myosin levels decreased, especially in the severely decompensated animals. One clear conclusion is that if a bladder is severely decompensated when reversals are performed, the bladder is incapable of significant recovery, as indicated by the contractility and collagen levels. Even if the smooth muscle recovers, it cannot overcome the inhibition of contraction mediated by the increased content and distribution of collagen . This has important connotations in men with severe obstructive dysfunction secondary to BPH. Pharmacological treatment at this level of dysfunction may be detrimental as severely decompensated bladders do not seem to improve, even if the duration of recovery is longer.
This material is based upon work supported in part by the Office of Research and Development Department of the Veterans Affairs, and in part by the Capital Region Medical Research Foundation.
Dr. Levin and his co-authors have nothing to disclose.
0.05% Tween 20 in TBS