In vivo hepatocyte growth factor gene transfer to bladder smooth muscle after bladder outlet obstruction in the rat: a morphometric analysis

PURPOSE: We determined whether hepatocyte growth factor gene transfer after partial bladder outlet obstruction would prove effective for decreasing transforming growth factor-beta expression and consequently decreasing collagen deposition in partially obstructed rat bladders. MATERIALS AND METHODS: Ten-week-old male Sprague-Dawley rats were divided into 3 groups of 10 each, including group 1--sham operation, group 2--bladder outlet obstruction for 4 weeks and group 3--hepatocyte growth factor gene transfer after bladder outlet obstruction. Two weeks after the onset of bladder outlet obstruction in group 3 hepatocyte growth factor-liposome complex (50 microg human hepatocyte growth factor cDNA) was injected into the smooth muscle of the rats. RESULTS: We noted no difference between groups 2 and 3 with regard to the ratio of bladder weight to body weight. The ratio in groups 2 and 3 was significantly higher than in group 1 (p = 0.043). The mean percent of collagen area +/- SE was 36.32% +/- 1.83%, 27.90% +/- 2.66% and 8.97% +/- 3.35% in groups 1 to 3, respectively (p <0.05). Relative hepatocyte growth factor and c-met mRNA and protein expression were higher in group 3 than in groups 1 and 2. However, the expression of transforming growth factor-beta1 mRNA and protein was higher in group 2 than in groups 1 and 3. CONCLUSIONS: These findings may imply a possible novel therapeutic strategy against bladder dysfunction arising in patients with bladder outlet obstruction.     

Relative bladder outlet obstruction

PURPOSE: Currently bladder outlet obstruction in males is defined by the provisional International Continence Society nomogram which is partly based on expert opinion and partly on measurements before and after transurethral prostate resection. Recently there has been some interest in the development of a similar nomogram for females. MATERIALS AND METHODS: We studied the possibility of defining bladder outlet obstruction based on a sign that it causes, namely post-void residual urine. RESULTS: The probability of relative post-void residual urine exceeding 20% of bladder capacity was modeled in males and females using 1 parameter, that is URA/w20 or the ratio of the obstruction parameter urethral resistance factor (URA)-to-the bladder contractility parameter Watts factor at 20% (w20). URA/w20 represents relative bladder outlet resistance or bladder outlet resistance normalized to bladder contractility. Above a threshold of URA/w20 = 6.8 in females and 8.2 in males a relative post-void residual exceeding 20% was noted in 90% of measurements. These thresholds may be used to define relative obstruction. The provisional International Continence Society nomogram for obstruction in males was transformed into an identical nomogram for females by equating the probabilities of post-void residual urine in each gender. The latter differed from that in men, in that the lines demarcating the zones were horizontal or flow rate independent but the intercepts were approximately the same at 20 and 40 cm. water. CONCLUSIONS: Instead of defining obstruction as an absolute level of bladder outlet resistance we suggest that it is better to define it relatively, that is as a level of bladder outlet resistance that depends on bladder contractility.     

Acute increase in blood flow to the rat bladder subsequent to partial bladder outlet obstruction

Partial obstruction of the rat bladder outlet initiates a multi-step process during which the bladder progressively loses its functional ability. The first step in this progression is bladder hypertrophy; the organ dramatically increases in size and weight to compensate for the effects of obstruction. Unoperated female rats, age-matched, sham-obstructed rats, and rats that received a partial bladder outlet obstruction were studied. During the first 24 hours after partial bladder outlet obstruction, relative bladder blood flow was measured using a fluorescent microsphere infusion technique and laser Doppler imaging. Nitric oxide synthase (NOS) activities of control and obstructed rat bladder tissues were determined using an enzymatic assay that measures the conversion of (3)H-L-arginine to (3)H-L-citrulline. Using the microsphere infusion technique, a significant increase in blood flow to the obstructed rat bladder was observed during the first 24 hours after partial bladder outlet obstruction. Relative bladder blood flow increased approximately sixfold at 4 and 6 hours post-obstruction and remained elevated through 24 hours of obstruction. Sham operations (evaluated after 6 hours after surgery) resulted in a minor increase in blood flow that did not reach statistical significance. Relative blood flow to the spleen, measured in the same rats, was not significantly changed. Laser Doppler measurements also identified a significant increase in rat bladder blood flow after outlet obstruction and showed that increased blood flow could be detected as early as 1 hour post-obstruction. Interestingly, despite the significant differences in bladder blood flow between control and early post-obstructed rat bladders, NOS activities of control and obstructed rat bladders were comparable. The increase in bladder blood flow precedes the urothelial, fibroblast and smooth muscle cell hyperplasia, and the smooth muscle hypertrophy that occurs after obstruction. We propose that, in response to surgical induction of partial outlet obstruction, acute up-regulation of bladder blood flow may be an initiating factor for subsequent bladder cell proliferation and smooth muscle hypertrophy. Neurourol. Urodynam. 19:195-208, 2000.     

Inducible nitric oxide synthase promotes pathophysiological consequences of experimental bladder outlet obstruction

PURPOSE: Bladder outlet obstruction leads to histological and functional changes in the bladder over time. We investigated the role of inducible nitric oxide synthase (iNOS) in the progression of pathological changes of the bladder secondary to outlet obstruction in a rat and a mouse model. MATERIALS AND METHODS: To assess expression of iNOS in the bladder, polymerase chain reaction amplification of mRNA was done. Rats were subjected to sham operation or partial bladder outlet obstruction. They were given the iNOS inhibitor aminoguanidine in drinking water or unmodified water. After 2 weeks, awake cystometric evaluation was performed, the bladders were harvested and the degree of fibrosis was assessed. In another series of experiments mice deficient in the iNOS gene (iNOS -/-) were compared to WT mice for cystometric as well as histological changes in the bladder following partial bladder outlet obstruction or sham operation. RESULTS: Partial bladder outlet obstruction induced the expression of iNOS mRNA in the mouse bladder. iNOS -/- mice showed a significantly smaller increase in bladder volume at 3 weeks compared with WT. Pharmacological inhibition of iNOS activity significantly attenuated the increase in bladder size and the number of spontaneous bladder contractions in obstructed rats at 2 weeks. Furthermore, genetic and pharmacological decreases in iNOS led to significantly less fibrosis of the bladder after partial bladder outlet obstruction in mice and rats, respectively. CONCLUSIONS: Pharmacological or genetic decreases in iNOS resulted in amelioration of functional and fibrotic changes in the bladder after partial bladder outlet obstruction, suggesting that NO contributes to the pathophysiology of bladder outlet obstruction.     

Involvement of angiotensin II type 1 receptor on pathological remodeling and dysfunction in obstructed bladder

Objectives: To determine whether long‐term administration of an angiotensin II type 1 receptor antagonist improves morphology and function in obstructed bladders. Methods: Male Sprague–Dawley rats underwent surgery to produce bladder outlet obstruction (bladder outlet obstruction group; n = 32) or sham surgery (sham group; n = 16). A total of 2 weeks later, 16 bladder outlet obstruction‐rats were given the AT1 antagonist, candesartan, subcutaneously (candesartan group) using an osmotic pump for 4 weeks; the remaining bladder outlet obstruction‐rats received vehicle (bladder outlet obstruction group). A total of 6 weeks after surgery, we compared continuous cystometry, bladder weight, strip contraction, histology and messenger ribonucleic acid expression of growth factors, nicotinamide adenine dinucleotide phosphate oxidase 1 and renin–angiotensin system components among the three groups. Results: Bladder weights markedly increased with bladder outlet obstruction (578 ± 159 mg), and candesartan (344 ± 111 mg) suppressed this increase. Micturition pressure, which was significantly higher with bladder outlet obstruction, was unaffected by candesartan. The shortened micturition interval and decreased micturition volume with bladder outlet obstruction were significantly prolonged and increased by candesartan. Candesartan also significantly decreased residual urine. Histologically, the collagen fiber‐to‐muscle ratio was significantly increased with bladder outlet obstruction (0.85 ± 0.25) compared with the sham group (0.53 ± 0.18); this increase was suppressed by candesartan (0.49 ± 0.21). The messenger ribonucleic acid expression of heparin‐binding epidermal growth factor‐like growth factor, transforming growth factor‐β1 and nicotinamide adenine dinucleotide phosphate oxidase 1 significantly increased with bladder outlet obstruction, but it was significantly reduced by candesartan. Compared with the bladder outlet obstruction group, candesartan increased the maximal contraction of bladder strips for all stimuli except for angiotensin II. Conclusion: These findings suggest that bladder angiotensin II type 1 receptors contribute to the pathophysiology of remodeling and dysfunction in obstructed bladder.     

In vitro rat bladder function after neonatal estrogenization

PURPOSE: Experiments were done to evaluate the functional effects of neonatal diethylstilbestrol (DES) treatment on bladder function in male and female Noble rats. MATERIALS AND METHODS: At 5 months after neonatal DES bladders were removed and weighed. Ventral and dorsal bladder strips were prepared to evaluate the effects of neonatal DES on contractile responses to electrical field stimulation, carbachol, adenosine triphosphate, phenylephrine and KCl. Relaxant responses to the catecholamines arterenol (norepinephrine), epinephrine and isoproterenol were also monitored. RESULTS: Neonatal DES resulted in significant increases in bladder mass in males and females. Contractile and relaxant responses were largely unchanged by neonatal DES treatment and the only change observed was a decreased response of ventral strips from male neonatal DES rats to 4 and 8 Hz. stimulation. Ventral strips from male control and neonatal DES rats responded to field stimulation and carbachol with significantly greater responses than dorsal strips and were more sensitive to the relaxant actions of norepinephrine and epinephrine. CONCLUSIONS: The data confirm that neonatal DES causes infravesical obstruction. However, in contrast to published reports of the effects of surgically induced mild outlet obstruction, neonatal DES treatment has little effect on in vitro bladder strip contractile or relaxant function. Thus, the neonatal DES treated rat does not seem to be a useful model in which to study the in vitro effects of partial outlet obstruction on the bladder.     

Bladder smooth muscle cell phenotypic changes and implication of expression of contractile proteins (especially caldesmon) in rats after partial outlet obstruction

BACKGROUND: The purpose of the present study was to investigate morphological changes in bladder smooth muscle of rats with partial outlet obstruction. We investigated smooth muscle cell phenotypic changes and implication of synthetic phenotype in contractility decrease and bladder compliance after bladder outlet obstruction. METHODS: Partial bladder outlet obstruction was introduced in female rats. Bladder were removed at 1, 3, 6, 10 and 20 weeks after the obstruction. Temporal pattern of changes in bladder mass, light microscopic pathogenesis and phenotypic expression of the bladder smooth muscle cells in the electron micrographs were investigated. Expression of contractile protein was also investigated by the immunoblotting method. RESULTS: Marked increase in bladder mass with marked thickening of smooth muscle layer was observed at 1 week after obstruction. The ratio of myocytes exhibiting contractile and synthetic phenotypes was almost constant until 6 weeks after the obstruction, but thereafter, synthetic phenotypes gradually increased and the ratio (synthetic/contractile phenotype) was 1.5-fold at 20 weeks after the obstruction. Caldesmon was most markedly expressed after the obstruction among contractile proteins examined by the immunoblotting method. CONCLUSION: Phenotypic changes were confirmed in bladder smooth muscle, and the decrease of the ratio of contractile phenotype was observed after long-term obstruction of the bladder outlet. Among the contractile proteins in the bladder smooth muscle cell, caldesmon was considered a reliable marker for predicting the pathogenetic conditions of the bladder.     

膀胱出口梗阻大鼠逼尿肌中神经生长因子mRNA的变化及其意义

目的探讨膀胱出口梗阻(BOO)大鼠模型膀胱逼尿肌中神经生长因子(NGF)mR- NA的表达变化及意义。方法建立BOO大鼠模型,采用逆转录-聚合酶链反应(RT-PER)检测大鼠膀胱逼尿肌中NGF mRNA的表达。结果:NGF mRNA在模型组和对照组的膀胱逼尿肌细胞中均有表达,模型组大鼠的逼尿肌NGF mRNA水平高于对照组,两组间的mRNA平均水平差异有统计学意义(P<0.01)。结论膀胱出口梗阻后,膀胱逼尿肌细胞中NGF mRNA表达水平增高,这可能与BOO所致膀胱功能受损的发生有关。     

Effects of partial bladder outlet obstruction and its relief on types I and III collagen and detrusor contractility in the rat

Bladder outlet obstruction induces a rapid hypertrophy characterized by increased bladder mass and collagen deposition. An increase in collagen is likely to reduce the contractility and compliance of bladder wall. This study was undertaken to investigate the effects of partial bladder outlet obstruction and its relief on types I and III collagen, and the relationship between detrusor contractility and collagen types. A total of 40 female rats was used for experiment and divided into one control, one obstruction, and three recovery groups. The contractility to field stimulation was recorded; total collagen and collagen concentration were quantified. The localization of types I and III collagen and the expression of pro-alpha1(I) and alpha1(III) collagen mRNA were determined by immunohistochemical staining and Northern blot hybridization, respectively. Contractile response to field stimulation was reduced after obstruction and recovered following relief. The total amount of collagen increased after obstruction and decreased after relief; however, collagen concentration decreased after obstruction and increased following relief. Contractility correlated negatively with total collagen but positively with collagen concentration. The protein deposition of types I and III collagen was localized in lamina propria and muscle bundles in all groups. The expression of types I and III collagen gene was up regulated after obstruction, but down regulated after relief. Negative correlation between contractility and gene expressions of collagen types was significant. These data suggest that the change in localization and quantity of collagen types leads to morphologic changes of bladder and can have an impact on the contractility of detrusor. Neurourol. Urodynam. 19:29-42, 2000.     

Inhibition of collagen deposit in obstructed rat bladder outlet by transplantation of superparamagnetic iron oxide-labeled human mesenchymal stem cells as monitored by molecular magnetic resonance imaging (MRI)

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly males. The present study is to investigate if human mesenchymal stem cells (MSCs) are capable of inhibiting collagen deposition and improve cystometric parameters in bladder outlet obstruction in rats. Human MSCs were labeled with nanoparticles containing superparamagnetic iron oxide (SPION), and transplanted in rat BOO lesion site. Forty 6-week-old female Sprague-Dawley rats were divided into four groups (group 1: control, group 2: sham operation, group 3: BOO, and group 4: BOO rats receiving SPION-hMSCs). Two weeks after the onset of BOO, 1 × 10(6) SPION-hMSCs were injected into the bladder wall. Serial T2-weighted MR images were taken immediately after transplantation of SPION-labeled human MSCs and at 4 weeks posttransplantation. T2-weighted MR images showed a clear hypointense signal induced by the SPION-labeled MSCs. While the expression of collagen and TGF-β protein increased after BOO, the expression of both returned to the original levels after MSC transplantation. Expression of HGF and c-met protein also increased in the group with MSC transplantation. Maximal voiding pressure and residual urine volume increased after BOO but they recovered after MSC transplantation. Human MSCs transplanted in rat BOO models inhibited the bladder fibrosis and mediated recovery of bladder dysfunction. Transplantation of MSC-based cell therapy could be a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.     

Calcineurin mediates bladder smooth muscle hypertrophy after bladder outlet obstruction

PURPOSE: Bladder outlet obstruction (BOO) leads to compensatory bladder hypertrophy. However, the hypertrophy mechanism remains elusive. We report that calcineurin (Cn) is involved in bladder hypertrophy. MATERIALS AND METHODS: Partial BOO was surgically induced in 10-week-old female Wistar rats. The bladder was excised 2, 4 and 6 weeks following surgery in 9 rats each. Histological study was performed at each time point. Cn expression was examined by Western blot analysis. Myosin heavy chain expression was evaluated on gels stained with Coomassie blue. Primary cultured bladder smooth muscle cells were infected with recombinant adenoviruses encoding a constitutive active form of CnA (DeltaCnA), CnB and lacZ, and cell size was measured. RESULTS: In histological findings bladder smooth muscle hypertrophy was observed 2 and 4 weeks after surgery. However, the thickened muscles became thinner 6 weeks after BOO. CnA expression 2 weeks after BOO increased 3.2-fold compared with that of controls. Expression significantly decreased 4 and 6 weeks after surgery. In contrast, CnB expression was unchanged throughout hypertrophy development. Changes in myosin heavy chain expression correlated with changes in CnA. We observed significant hypertrophy in DeltaCnA and CnB over expressing smooth muscle cells. Moreover, FK506, which is a potent inhibitor of Cn, blocked hypertrophy in DeltaCnA and CnB over expressing smooth muscle cells. CONCLUSIONS: These data suggest that Cn has an important role in the induction of bladder smooth muscle hypertrophy.     

Downregulation of TWIK‐related arachidonic acid‐activated K+ channel in the spinal cord of rats after complete bladder outlet obstruction

Objectives: To study the altered expression of TWIK‐related arachidonic acid‐activated K⁺ channel in the L6–S1 spinal cord of rats after complete bladder outlet obstruction, and to investigate the role of TWIK‐related arachidonic acid‐activated K⁺ channel in the neurogenic mechanism of bladder dysfunction. Methods: Female Sprague–Dawley rats were randomly divided into a complete bladder outlet obstruction group and a sham‐operated control group. Cystometry was carried out and tissues of L6–S1 spinal cord were obtained for detection of TWIK‐related arachidonic acid‐activated K⁺ channel mRNA and protein by real‐time polymerase chain reaction, western blot and immunohistochemistry. Results: The bladder outlet obstruction rat model was established. Real‐time polymerase chain reaction, western blot and immunohistochemistry showed that the expression of TWIK‐related arachidonic acid‐activated K⁺ channel was lower in the L6–S1 spinal cord of the bladder outlet obstruction rats, compared with the control rats. Conclusions: Downregulation of TWIK‐related arachidonic acid‐activated K⁺ channel might enhance the excitability of the neurons and increase the sensitivity of the bladder, probably providing a new study model of overactive bladder secondary to bladder outlet obstruction.     

Effects of outlet obstruction on glucose metabolism of the rabbit urinary bladder

Bladder outlet obstruction has been shown to cause detrusor contractile dysfunction. To determine if alterations in bladder metabolism may in part underlie these functional defects, we investigated the effects of mild outlet obstruction on the glucose metabolism of the rabbit urinary bladder. Mild outlet obstruction was created in mature male rabbits by the surgical placement of a silicon sleeve around the bladder neck. Two weeks after surgery, the in vitro ability of the obstructed bladder tissues to metabolize glucose was compared to that of the controls. The results can be summarized as follows: 1) The bladder wet weight increased 2.3-fold following two weeks of obstruction. 2) Obstructed bladder tissues had a reduced glucose consumption as compared to the controls. 3) CO2 generation was significantly reduced by 31% in obstructed bladder tissues whereas lactate formation increased significantly by 22%. 4) Tissue concentrations of ATP, creatine phosphate, and glycogen before incubation showed no significant differences between control and obstructed bladder tissues. In summary, bladder tissues following two weeks obstruction showed a decrease in aerobic metabolism and an increase in anaerobic metabolism. Previous studies have indicated that the ability of the bladder to maintain a contraction and empty may be directly related to aerobic metabolism. Therefore, the decrease in aerobic metabolism (even in the presence of increased anaerobic metabolism) may in part explain the decreased ability of the obstructed bladder to empty.