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Keywords:

  • bladder;
  • free radicals;
  • obstruction;
  • oxidative stress;
  • rabbit

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Objectives

Oxidative stress is a major etiology of obstructed bladder dysfunction. The major goal of the current study was to correlate the level of oxidative stress with both the severity and duration of obstruction.

Methods

A total of 32 New Zealand White rabbits were divided into four equal groups. Groups 1–3 received partial bladder outlet obstructions by standard methods and survived for 4, 8 or 12 weeks. Group 4 received sham surgery at the end of each time period, isolated strips were taken for contractility studies and the balance of the bladder was frozen as muscle and mucosa for quantification of nitrotyrosine and carbonyl-oxidized proteins derivatized into dinitrophenyl. For each duration, the eight rabbits were divided into three severity groups: mild, intermediate or severe decompensation.

Results

Contractile responses decreased in proportion to both severity and duration. The level of both oxidative products correlated to a much higher degree with the level of severity than the duration. There were significant decreases in the contractile responses in the mild decompensation group, whereas the level of derivatized into dinitrophenyl and nitrotyrosine of the muscle remained at control levels. This was not the case for the 4 weeks obstructed group.

Conclusions

These findings suggest that the etiology for the mechanism of contractile dysfunction is not an oxidative stress.


Abbreviations & Acronyms
ATP

adenosine triphosphate

BCA

bicinchoninic acid

BPH

benign prostatic hyperplasia

DNP

dinitrophenol

FS

field stimulation

IACUC

Institutional Animal Care and Use Committee

KCl

potassium chloride

NT

nitrotyrosine

RNS

reactive nitrogen species

ROS

reactive oxygen species

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

The function of the urinary bladder is to collect and store urine at low intravesical pressure and then, periodically, to expel the urine by a highly coordinated and sustained contraction.[1, 2] Bladder function depends on several factors including the state of innervation, vascularization, structure of the organ as a whole, contractile response of the smooth muscle elements to autonomic stimulation, availability of metabolic energy (cytosolic ATP and mitochondrial oxidative metabolites), and the density and distribution of connective tissue in the detrusor.[1, 3-5] These factors are intimately associated, and an alteration in one factor can induce substantial adaptive changes in the others.

Urinary bladder outlet obstruction is a common medical problem: more than 80% of males aged older than 50 years have varying degrees of bladder outlet obstruction secondary to BPH.[6-8] In humans, it is difficult to investigate the cellular mechanisms by which progressive bladder dysfunction occurs secondary to BPH. However, many of the functional changes associated with human-bladder pathology can be induced in experimental animal models, including the rabbit.[4, 9, 10] Rabbit bladder capacity is between 50 and 100 mL; compliance can be evaluated cystometrically using an 8-Fr Foley catheter to catheterize the bladder. The cystometric curve of the rabbit is similar in shape to that of humans: the bladder fills at low intravesical pressure until capacity is reached, at which time the pressure rises sharply. Similar to humans, bladder emptying occurs during the tonic phase of contraction. The bladder's ability to sustain increased pressure in response to stimulation is significantly reduced by partial outlet obstruction before any change in maximal-pressure generation occurs. This decreased ability to sustain increased pressure during stimulation is the reason that, in rabbits and humans, the bladder's ability to empty is reduced at times when the organ is capable of maximal-pressure generation.

Major characteristics of the rabbit's response to partial outlet obstruction; that is, an increase in bladder mass to a stable level, reduced compliance during bladder filling and development of overactive bladder syndrome (unstable bladder contractions during filling) in ∼30% of obstructed animals,[4, 10, 11] are similar to those secondary to BPH in men. Ultrasound studies have confirmed that not only do men with obstructive uropathies show an increase in bladder mass,[12-15] but bladder wall thickness has been shown to be the most accurate non-invasive way to identify men with obstructive bladder dysfunction.[16-18]

Results of experiments in which mild partial outlet obstruction was studied longitudinally (up to 6 months) showed that the level of bladder decompensation was related to the magnitude of the increase in bladder mass, but not to duration of obstruction.[19, 20]

Although it is clearly true that the longer rabbits are obstructed, the greater proportion of them shifts to decompensation and progress to severe decompensation, individual rabbits might remain compensated for prolonged periods of time.

Elevations in ROS and RNS, and changes in the activity of anti-oxidant mechanisms are induced by partial outlet obstruction and in vivo ischemia/reperfusion.[21-27] Oxidative injury occurs when ROS and RNS react with endogenous macromolecules (lipids, proteins and nucleic acids) and disrupt normal cellular function. Reaction of specific ROS and RNS with lipids, proteins and nucleic acids results in the formation of characteristic products that can be used to act as biomarkers for oxidative stress. Two such biomarkers are nitrotyrosine for RNS free radicals and carbonylated proteins. Nitrotyrosine is a product of tyrosine nitration mediated by ROS species of free radicals. It has been found to be associated with a large number of pathological conditions including diabetes.[28, 29] Carbonylated proteins can be quantitated using western analyses for DNP (a derivatized product of carbonylated proteins). Similar to nitrotyrosine, ROS-mediated protein oxidation has been linked to a variety of diseases including heart disease,[30] Alzheimer's disease and other neurological diseases.[31, 32]

The specific aim of the present study was to determine if the level of oxidative stress induced by partial outlet obstruction in rabbits relates more to duration of obstruction or severity of obstruction.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

All methods utilized in these experiments were approved by the IACUC Committee of the Stratton VA Medical Center.

A total of 32 New Zealand White rabbits were divided into four equal groups. Groups 1–3 received partial outlet obstructions by surgically placing a silk ligature loosely around the catheterized urethra of an anesthetized rabbit. The rabbits from each group were allowed to recover for 4, 8 or 12 weeks. The rabbits in group four received sham surgery and two rabbits were allowed to recover from sham surgery for 4 weeks; and three rabbits were allowed to recover for each of 8 and 12 weeks. There were no significant differences in bladder function in the sham group and the data from these eight rabbits were combined. It should be mentioned that a single person with 14 years experience with this surgical method carried out all surgeries, thus insuring consistency in the surgical methodology.

At the end of each time period, the rabbits were anesthetized with isoflorane, the bladders excised and weighed, and three isolated full thickness strips were taken for contractility studies. The balance of the bladders were separated by blunt dissection into bladder smooth muscle and mucosa, frozen in liquid nitrogen and stored at −80°C for biochemical quantification of NT and DNP as markers for oxidative stress by western analyses.

In regard to the contractility studies,[33] each isolated strip was mounted in individual 15-mL baths containing warmed oxygenated Tyrodes solution and allowed to equilibrate for 30 min. A passive length–tension curve, which is a measure of the compliance of the strip (mg of tension per mm of length), was generated and contractile responses (maximal tension generation [mg tension/cross-sectional area] and maximal rate of tension generation [mg tension generated/s]) to FS (32 Hz) were used to determine the passive length that allows for maximal active tension generation. At this length, the responses to FS at 2, 8 and 32 Hz, ATP (1 mmol/L), carbachol (20 μmol/L) and KCl (120 mmol/L) were determined. Each strip was washed three times at 15 min intervals with fresh warmed oxygenated buffer between pharmacological additions.

We found that the rate of tension generation to FS is the most sensitive measure of contractile dysfunction and sensitivity to drugs; thus we determined the maximal tension and rate-of-tension development for field stimulation at all frequencies of stimulation.[34-36] All data were recorded on a Grass model D polygraph (Grass Technologies, Astro-Med Industrial Park, West Warwick, RI, USA) and the data were digitized and analyzed using the Polyview system of A-D conversion (Grass Technologies, Astro-Med Industrial Park). It should be noted that in our experience, carrying out dose–response curves for all agents would lead to fatigue because of the duration of each experiment, especially in the obstructed bladder strips, thus making the data unreliable.

In regard to the western blot assays for NT and DNP,[25, 26] frozen tissues of bladder muscle wall and mucosa were homogenized on ice in homogenization buffer (50 mmol/L Tris, pH 7.5, 5% Tiron) containing the Halt Protease inhibitor Cocktail (Pierce, Rockford, IL, USA) at 100 mg/mL. After addition of sodium dodecylsulfate (final concentration, 1%), the sample was boiled for 4 min and centrifuged at 4400 g for 15 min. Protein concentration in the supernatant was measured using the Pierce BCA (bicinchoninic acid) protein assay kit (Pierce). Membranes were blocked with 5% non-fat milk in 0.05% Tween 20 in phosphate-buffered solution for 1 h at room temperature and then incubated with primary antibody, monoclonal antibody to nitrotyrosine (Alexis Biochemicals, San Diego, CA, USA) and anti-2,4-dinitrophenol (DNP; Bethyl Laboratories, Montgomery, TX, USA). After treatment with the primary antibody, the membranes were washed and incubated with secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The substrates were visualized by using ECL-Plus (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 2 min, and analyzed with a Kodak Image Station 440CF and Kodak 1D image analysis software (Scientific Image System, Rochester, NY, USA).

Decompensation categories were determined by bladder weight: under 6 g were mild, 6–20 g were intermediate and over 20 g were severe. This categorization is used so that when we carry out reversal experiments, at the time of reversal we can calculate the bladder weight by measuring the mass of the empty bladder in vivo using calipers measuring length, vertical and horizontal width. Thus, using bladder weight to categorize the severity of the obstruction, we can categorize the bladder severity at the time of reversal without surgical intervention.[37]

The statistical methods utilized one-way anova followed by the Tukey test for individual differences among the groups.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Figure 1a shows the bladder weight as a function of duration. There is a clear progressive increase in bladder weight. Figure 1b shows the distribution of mild, intermediate and severe decompensation among the three durations of obstruction. After 4 weeks of obstruction, there were three out of eight mild, three out of eight intermediate and two out of eight severe; after 8 weeks there were two out of eight mild, four out of eight intermediate and two out of eight severe; while after 12 weeks obstruction there were no mild, four out of eight intermediate and four out of eight severe.

figure

Figure 1. (a) The bladder weight as a function of duration. (b) The distribution of mild, intermediate and severe decompensation among the three durations of obstruction. †Significantly different from control. ‡Significantly different from all other durations, P < 0.05. (a) image, Control; image, obstructed 4 weeks; image, obstructed 8 weeks; image, obstructed 12 weeks; (b) image, obstructed 4 weeks; image, obstructed 8 weeks; image, obstructed 12 Weeks.

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Figure 2a shows the maximal contraction to field stimulation sorted by duration, and Figure 2b shows the same as sorted by severity. In both cases, the responses of all bladders were significantly lower than the response of the control bladder. In regard to duration, the responses of the 8 and 12 weeks obstructed bladders were significantly lower than both control and 4 weeks obstructed. In regard to severity, the responses of the intermediate and severe decompensated bladders were significantly lower than both control and mild groups.

figure

Figure 2. (a) The maximal contraction to field stimulation sorted by duration. †Significantly different from control. ‡Significantly different from control and 4 weeks obstructed, P < 0.05. (b) The maximal contraction to field stimulation sorted by severity. †Significantly different from control; ‡Significantly different from control and mild obstruction, P < 0.05. (a) image, Control; image, obstructed 4 weeks; image, obstructed 8 weeks; image, obstructed 12 weeks; (b) image, control; image, mild obstruction; image, intermediate obstruction; image, severe obstruction.

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Figure 3a shows the maximal rate of contraction to field stimulation sorted by duration, and Figure 3b shows the same as sorted by severity. In both cases, the responses of all bladders were significantly lower than the response of the control bladder. In regard to duration, the responses of the 8 and 12 weeks obstructed bladders were significantly lower than both control and 4 weeks obstructed. In regard to severity, the responses of the intermediate and severe decompensated bladders were significantly lower than both control and mild groups.

figure

Figure 3. (a) The maximal rate of contraction to field stimulation sorted by duration. †Significantly different from control. ‡Significantly different from control and 4 weeks obstructed, P < 0.05. (b) The maximal rate of contraction to field stimulation sorted by severity. †Significantly different from control. ‡Significantly different from control and mild obstructed, P < 0.05. (a) image, Control; image, obstructed 4 weeks; image, obstructed 8 weeks; image, obstructed 12 weeks; (b) image, control; image, mild obstruction; image, intermediate obstruction; image, severe obstruction.

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Figure 4a shows the maximal responses to carbachol, KCl and ATP sorted by duration, and Figure 3b shows the same as sorted by severity. In both cases, the responses of all bladders to all forms of stimulation were significantly lower than the responses of the control bladder. In regard to duration, the responses of the 8 and 12 weeks obstructed bladders were significantly lower than both control and 4 weeks obstructed. In regard to severity, the responses of the intermediate and severe decompensated bladders were significantly lower than both control and mild groups.

figure

Figure 4. (a) The maximal responses to carbachol, KCl and ATP sorted by duration. †Significantly different from control. ‡Significantly different from control and 4 weeks obstructed, P < 0.05. (b) The maximal responses to carbachol, KCl and ATP sorted by severity. †Significantly different from control. ‡Significantly different from control and mild obstruction, P < 0.05. (a) image, Control; image, obstructed 4 weeks; image, obstructed 8 weeks; image, obstructed 12 weeks; (b) image, control; image, mild obstruction; image, intermediate obstruction; image, severe obstruction.

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Figure 5a shows the concentration of NT in the muscle sorted by duration, and Figure 5b shows the same as sorted by severity. In regard to duration, the concentration of NT of 4 and 8 weeks obstructed were significantly above the control, and the concentration in the 12 weeks obstructed was significantly greater than all groups. In regard to severity, the concentration of NT in the mild group was no different from the control group, whereas there was a progressive increase in NT concentration for the intermediate and severe groups.

figure

Figure 5. (a) The concentration of NT in the muscle sorted by duration. (b) The concentration of NT in the muscle as sorted by severity. †Significantly different from control. ‡Significantly different from all other conditions, P < 0.05.

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Figure 6a shows the concentration of NT in the mucosa sorted by duration, and Figure 6b shows the same as sorted by severity. In regard to duration, the concentration of NT of 4, 8 and 12 weeks obstructed were significantly above the control, but no different from each other. In regard to severity, the concentration of NT in the mild, intermediate and severe groups were all significantly increased compared with the control, but no different from each other.

figure

Figure 6. (a) The concentration of NT in the mucosa sorted by duration. †Significantly different from control, P < 0.05. (b) The concentration of NT in the mucosa sorted by severity. †Significantly different from control, P < 0.05.

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Figure 7a shows the concentration of DNP in the muscle sorted by duration, and Figure 7b shows the same as sorted by severity. In regard to duration, the concentrations of DNP of 4 and 8 weeks obstructed were significantly above the control, and the concentration in the 12 weeks obstructed was significantly greater than all groups. In regard to severity, the concentration of DNP in the mild group was no different from the control, whereas there was a progressive increase in DNP concentration for the intermediate and severe groups.

figure

Figure 7. (a) The concentration of DNP in the muscle sorted by duration. †Significantly different from control. ‡Significantly different from all other conditions, P < 0.05. (b) The concentration of DNP in the muscle sorted by severity. †Significantly different from control, P < 0.05.

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Figure 8a shows the concentration of DNP in the mucosa sorted by duration, and Figure 8b shows the same as sorted by severity. In regard to duration, the concentration of DNP of 4, 8 and 12 weeks obstructed were significantly above the control. In regard to severity, the concentration of DNP in the mild group was no different from the control, whereas there was a progressive increase in DNP concentration for the intermediate and severe groups.

figure

Figure 8. (a) The concentration of DNP in the mucosa sorted by duration. †Significantly different from control, P < 0.05. (b) The concentration of DNP in the mucosa sorted by severity. †Significantly different from control, P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

The progression of obstructive bladder dysfunction in both men and in rabbits occurs at different rates for different individuals. Some men and rabbits can remain in a compensated obstructed state for prolonged periods of time, whereas others can progress to severe decompensation within a relatively short time period.[2, 19, 20, 38] In both cases, oxidative stress (free radical generation and damage) is one of the major etiologies of obstructive damage for both men and rabbits.[11, 22, 23, 39]

The specific aim of the present study was to determine the relationship between the duration of partial outlet obstruction in rabbits and the severity of bladder dysfunction with the level of oxidative stress. The following schematic is how we view the progression of obstructive bladder dysfunction:

Thumbnail image of

There are two major pathways that can result in bladder dysfunction. The first is the generation of free radicals and subsequent oxidative damage to cellular and subcellular structures, such as the mitochondria, sarcoplasmic reticulum and synaptic membranes.[21, 22, 39]

The second pathway relates to calcium-overload, which is the increase in cytosolic-free calcium that occurs during bladder ischemia. Increased cytosolic-free calcium can activate a variety of degradative enzymes including calpain and phospholipase A2, which itself can result in cellular and subcellular damage.[40-44]

Interestingly, when we analyzed the oxidative stress by severity of bladder dysfunction, there was no increase in either muscle NT or DNP, although there was a significant decrease in the contractile responses to all forms of stimulation. This would indicate that oxidative stress might not be a major factor in the decreased contractile responses in the mild obstructive group.

In general, for the bladder smooth muscle, the level of oxidative stress is definitely a function of the severity of the dysfunction rather than the duration. Although, it is certainly true that the longer the rabbit is obstructed, the more rabbits progress to a more dysfunctional state and have a greater level of oxidative stress.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References
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