Free radical damage as a biomarker of bladder dysfunction after partial outlet obstruction and reversal.

OBJECTIVE: To investigate the use of free-radical generation as a result of protein carbonylation and nitrotyrosination to characterize the level of bladder dysfunction after partial bladder outlet obstruction (PBOO) and reversal. MATERIALS AND METHODS: We surgically created PBOO in male New Zealand White rabbits; after 4 weeks of PBOO, one group of six rabbits was assessed, while the PBOO was relieved in two additional groups of six rabbits each that were assessed at 4 and 8 weeks after relieving the PBOO. Six sham-operated rabbits served as controls. Sedated rabbits were assessed by cystometry and the bladders were then removed for contractile, histological and molecular studies. Western blotting was used to determine the level of carbonylation and nitrotyrosination at the protein level. RESULTS: The PBOO group had significant decreases in the contractile responses to field stimulation, ATP, carbachol and KCl. The responses to all forms of stimulation increased significantly at 4 weeks after reversal, and further increased to near normal levels by 8 weeks. Similarly, compliance and cystometric values also returned to near normal values after reversal. The hypertrophied smooth muscle of the obstructed bladders regressed to near-normal size. There was a significant increase in the level of carbonylation and nitrotyrosination after PBOO, and a progressive decrease in the 4-week reversal groups, nearing control values by 8 weeks. CONCLUSIONS: Significantly increased carbonylation and nitrotyrosination levels after PBOO correlated with the severe dysfunction in the obstructed rabbits. Similarly, decreased levels of oxidation and nitration correlated with the functional recovery after reversal.

Changes in the smooth muscle of the corpora cavernosum related to reversal of partial bladder outlet obstruction in rabbits.

Previous studies have demonstrated that partial bladder outlet obstruction (PBOO) in the rabbit induces an increase in corpus cavernosum smooth muscle (CCSM) tone, which may make it difficult for the CCSM to relax. Thus, to determine whether the corpus cavernosum restores relaxation after reversal of PBOO, we investigated the physiologic, histologic, and cell biology in penises obtained from rabbits 4 weeks and 8 weeks after reversal of PBOO. CCSM from bladder outlet-obstructed and obstruction-reversed rabbits showed significant decreases in the contractile responses to phenylephrine. The relaxation responses to electrical field stimulation (EFS), ATP, acetylcholine, and sodium nitroprusside (SNP) were decreased in obstructed and reversed for 4 weeks groups. By 8 weeks of reversal, the relaxation of CCSM was increased gradually in response to EFS, SNP, and acetylcholine. However, the response to ATP did not result in the relaxation of CCSM to control levels. The ratio of SM to collagen decreased after obstruction and remained low after reversal. Expression of both isoforms of Rho kinase (ROK) was increased in obstruction groups. At 4 weeks of reversal, the expression of ROK alpha remained at obstruction level, whereas ROK beta expression decreased in comparison with the obstruction group. By 8 weeks of reversal, expression of both ROK alpha and beta significantly decreased when compared with the obstruction group. These results suggested that the poor relaxation response at reversal of 4 weeks was associated with incomplete decreased expression of both isoforms of ROK, whereas the incomplete recovery of the CCSM relaxation response at reversal of 8 weeks may be associated with structural alterations in the CC and irreversible damage from PBOO.

The effect of chronic partial bladder outlet obstruction on corpus cavernosum smooth muscle and Rho-kinase in rabbits.

AIM: The goal of this study was to investigate the effect of different severities in bladder dysfunction on corpus cavernosum physiology, morphology and expression of Rho-kinase in rabbits. METHODS: Male New Zealand rabbits were divided into control, 2 and 8 weeks of partial bladder outlet obstruction (PBOO) groups. Isolated cavernosal strips from all groups were precontracted with phenylephrine and the relaxant responses to electrical field stimulation (EFS), ATP, acetylcholine, and sodium nitroprusside (SNP) were determined. Histological and molecular studies were performed. RESULTS: Corpus cavernosum smooth muscle (CCSM) from 8 weeks obstruction rabbits showed significant decreases in the contractile response to phenylephrine and further decreased relaxation responses to EFS in comparison to 2 weeks group. Relaxation induced by ATP, acetylcholine, and SNP were all significantly diminished at both 2 and 8 weeks obstruction equally. The ratio of smooth muscle to collagen decreased at 2 weeks and further dropped at 8 weeks obstruction. Expression of both isoforms of Rho-kinase were increased in both obstruction groups at 2 weeks obstruction and decreased significantly (from the 2 week obstructed values) at 8 weeks while remaining above control values. CONCLUSION: The present study indicated that severe bladder dysfunction secondary to chronic PBOO induced significant physiological dysfunctions of CCSM as well as morphological changes. Activities of both ROK isoenzymes showed increases at 2- and 8-week obstructions. Increase in Rho-kinase expression/activity would be expected to make the CCSM more difficult to relax and also contribute to reduction of EFS-induced relaxation of CCSM after chronic PBOO.

Expression of parathyroid-hormone- related protein in the partially obstructed rabbit bladder.

INTRODUCTION: Parathyroid-hormone-related protein (PTHrP) is considered as an autocrine/paracrine regulator of growth and/or differentiation in normal and malignant tissues. We determined the distribution and density of the expression of PTHrP in the rabbit bladder during growth in response to partial outlet obstruction and its relation with the smooth muscle/collagen ratio. MATERIALS AND METHODS: A total of 30 male New Zealand White rabbits were studied. After 7, 14, 28 or 56 days of obstruction, 6 rabbits per group were sacrificed and the bladder extracted. Six sham-operated rabbits served as controls. The expression and localization of PTHrP or collagen type III were detected by immunohistochemistry using specific antibodies. RESULTS: The levels of PTHrP were progressively increased by 14 days of obstruction, whereas they decreased after 14 days, although the PTHrP positivity was higher than in sham controls by 28 and 56 days of obstruction. PTHrP staining was related to the smooth muscle/collagen ratio, both showing a peak in rabbits obstructed for 14 days. CONCLUSIONS: These data indicate that PTHrP may play an important regulatory role in the cellular and vascular response to partial outlet obstruction.

Effect of letrozole on urinary bladder function in the female rabbit.

OBJECTIVE: To investigate the effect of letrozole (a potent aromatase inhibitor that effectively inhibit the synthesis of oestrogen) on bladder contraction with changes in morphology and biochemistry. MATERIALS AND METHODS: Sixteen female New Zealand white rabbits were separated into four equal groups; groups 1-3 were given oral letrozole for 1, 2 and 3 weeks, and group 4 was given saline and served as the control group. At the end of the medication period each rabbit was anaesthetized and the bladder muscle strips were used for contractile, histological and biochemical studies. RESULTS: The concentration of serum oestrogen was significantly lower and testosterone was significantly higher in letrozole-treated rabbits than in the control group. The rabbits treated for 1 week with letrozole showed significant decreases in the contractile responses to electrical field stimulation, ATP and carbachol, but not to KCl. Contractility returned to normal in the rabbits treated for 2 and 3 weeks. Letrozole resulted in an increased volume percentage of collagens and decreased bladder compliance. The volume percentage of the smooth muscle component also changed, with a significant decrease at 1 week and then a gradual increase at 2 and 3 weeks. Contractile dysfunction was absent at 2 and 3 weeks, which was consistent with no change in sarcoplasmic reticulum Ca(2+)-ATPase content or mitochondrial function. CONCLUSIONS: The bladder contractility decline in the first week and was restored at 2 and 3 weeks. The present study unexpectedly showed the possibility that testosterone might be as important as oestrogen in the contractile function of the female bladder.

Estrogen induces angiogenesis of the female rabbit bladder.

Postmenopausal bladder dysfunction has been speculated to involve decreased circulating estrogen levels. It is our hypothesis that estrogen induces bladder dysfunctions by modulating blood flow to the bladder, i.e. low estrogen reduces blood flow to the bladder, whereas high estrogen increases blood flow. Our previous studies have demonstrated that estrogen administration in female rabbits induces a 'functional hypertrophy' of the urinary bladder smooth muscle represented by increased smooth muscle mass, which corresponds to increased contractile responses to all forms of stimulation. The present study investigates the effect of estrogen on vasculature density and distribution. Twenty-four female New Zealand white rabbits were separated into six groups of four rabbits each. Group 1 served as controls. Groups 2-6 were ovariectomized. Two weeks after ovariectomy (Ovx), groups 3-6 were given 17-beta estradiol (1 mg/kg per day) by s.c. implant for 1, 3, 7, and 14 days respectively. Blood vessel density and distribution were evaluated by immunohistochemistry and quantitative image analyses. Ovx resulted in significant vascular degeneration and decreased density, whereas estradiol administration mediated a significant angiogenic effect characterized by increased vascular density, and distribution of new vasculature within the smooth muscle bundles of the detrusor. Estradiol-induced vasculogenesis corresponds with our previously demonstrated increase in blood flow to the bladder and increased contractility. The most interesting aspect of these studies is the increased vascularization localized within the muscle bundles rather than between the muscle bundles, which may be important in the link between estrogen and increased incidence of cancers.

Role of angiogenesis in bladder response to partial outlet obstruction.

Benign prostatic hyperplasia (BPH) is a disease that has its etiology in the abnormal growth of the adult human prostate gland that accompanies the aging process in men. The symptomatic presentation of this disease, however, is related largely to degenerative changes in the bladder that occur as a result of the increasing urethral resistance and partial bladder outlet obstruction (PBOO) caused by the growing prostate gland. BPH is characterized by bladder hypertrophy, significant decreases in urinary flow and compliance, presence of residual urine after voiding, voiding urgency and incontinence (). Obstructed bladder dysfunction secondary to BPH is a slow, progressive disease that is so strongly associated with human aging that it is an expected occurrence of the male aging process. Although the symptoms of BPH are usually not life threatening, they effect an extremely negative quality of life for men who suffer from them. However, many men delay seeking medical treatment for early BPH since bladder function can remain relatively normal as the hypertrophying bladder initially compensates for the progressive increase in urethral resistance caused by prostatic obstruction. The limited changes in micturition pressure and flow characteristics that occur during compensated function are not usually disabling enough to motivate seeking medical attention, which, often, is not sought until the symptoms become typical of advanced disease. Recent advances in detection methods enable identification of patients with significant BPH during compensation before the bladder becomes dysfunctional (decompensated). A more complete understanding of the disease processes that underlie the loss of bladder function associated with BPH might enable the development of treatments that better protect these early-stage BPH patients from the more debilitating aspects of the disease. This review updates the understanding of obstructive bladder dysfunction via the use of animal models.

The effect of low-dose estrogen therapy on ovariectomized female rabbit bladder.

OBJECTIVES: To determine whether low-dose estrogen supplementation is as effective as high-dose supplementation in increasing bladder contractile function and mediating bladder hypertrophy and angiogenesis. METHODS: Sixteen New Zealand white female rabbits were separated into four groups of 4 rabbits each. Group 1 served as the control, and groups 2 to 4 underwent ovariectomy. The group 2 rabbits were studied 7 days after ovariectomy. The rabbits in groups 3 and 4 were medicated with 17-beta estradiol at a dose of 0.1 mg/kg/day and 1.0 mg/kg/day, respectively, for 7 days. At the end of the experiment each rabbit was anesthetized and the bladder removed for contractile, morphologic, and biochemical studies. RESULTS: Low- and high-dose estrogen administration resulted in similarly significant increases in the contractile responses to field stimulation, adenosine triphosphate, and potassium chloride. Similarly, both doses of estrogen mediated significant hypertrophy of the smooth muscle and decrease in collagen, similar levels of angiogenesis, and similar increases of citrate synthase activity. CONCLUSIONS: Low-dose estrogen produces similar physiologic, morphologic, and biochemical effects on the bladder as have been shown for high-dose estrogen.

Role of nitric oxide in urinary bladder function: effect of L-arginine.

BACKGROUND: Evidence indicates that decreased blood flow to the bladder plays a major role in obstructive bladder dysfunction in the rabbit model of partial bladder outlet obstruction (PBOO), and that nitric oxide (NO) regulation of blood flow may be important in modulating the degree of obstructive bladder dysfunction. The specific aim of our study is to determine the effect of feeding rabbits a diet high in L-arginine on the response to PBOO. MATERIALS AND METHODS: Sixteen male NZ White rabbits were separated into 4 groups of 4 each. The rabbits in groups 1 and 3 underwent PBOO. The rabbits in groups 2 and 4 were sham-operated. For 1 week prior to surgery, and 2 weeks postoperatively, each rabbit in groups 1 and 2 was put on a diet containing 7% arginine. Rabbits in groups 3 and 4 were on a normal diet (0.76% arginine). RESULTS: PBOO resulted in a greater increase in bladder weight in the control group than the arginine group. PBOO resulted in a greater decrease in compliance in the control group than the arginine group. The contractile responses to all agents in the arginine control group were greater than in the control normal diet group. PBOO resulted in a greater decrease in the response to field stimulation in the control group than in the arginine group. CONCLUSIONS: These studies clearly demonstrate that feeding rabbits a diet high in L-arginine was beneficial for the control rabbits, and reduced the level of dysfunctions following PBOO.

Effect of age on the response to short-term partial bladder outlet obstruction in the rabbit.

OBJECTIVE: To compare the physiological and structural changes after short-term partial bladder outlet obstruction (PBOO) in young and old rabbits, as PBOO results in marked contractile and histological alterations in the bladder. MATERIALS AND METHODS: In all, 20 young (7-8-week-old) and 20 old (2 years old) male rabbits were divided into four subgroups of five each (four obstructed and one sham control rabbit). The rabbits in the groups were evaluated after 1, 3, 7 and 14 days of PBOO, respectively. At the end of the respective periods, cystometry and contractile responses to field stimulation (FS), ATP, carbachol and potassium chloride were determined. Full-thickness sections of the bladder body and base were used to determine the vascular density, nerve density and smooth muscle/collagen ratios. RESULTS: The bladder weight of young rabbits increased at 1-7 days of PBOO and returned toward control levels at 14 days of PBOO, while in old rabbits it was higher than the control during the entire experiment. For the young rabbits, the responses to field stimulation decreased progressively for 1, 3 and 7 days, and increased significantly at 14 days. For old rabbits there was a progressive decrease to a minimal response by 3 days of PBOO and the response remained at this level over 14 days. The contractile response to ATP, carbachol and KCl were similar to the responses to FS. The vascular density in both groups increased to a maximum at 7 days and then decreased toward control values at 14 days. For the young rabbits, nerve density decreased more than in old rabbits. In the old group, the smooth muscle/collagen ratio was increased throughout PBOO and was higher than in young rabbits. The connective tissue compartment was markedly greater than in the young rabbits and the basal mucosa had vacuoles which were not apparent in the young bladders. CONCLUSIONS: This study shows that the adaptive changes to PBOO are faster in young rabbit bladders than in older rabbits.

Long term partial bladder outlet obstruction induced contractile dysfunction in male rabbits: a role for Rho-kinase.

AIMS: In this study we examined the expression of Rho-kinase (ROK) isoforms in rabbit detrusor smooth muscle during the progression of partial bladder outlet obstruction and correlated them with the time course of obstruction. METHODS: Detrusor samples were obtained from bladders after 1, 2, 4, and 8 weeks of obstruction and also sham operated control rabbits. Contractile responses to field stimulation (FS) and also the smooth muscle (SM) to collagen ratio were determined in isolated bladder strips. Reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were used to determine the relative levels of ROK isoform expression at the mRNA and protein levels. RESULTS: Bladder weight increased gradually and contractile responses were reduced significantly over the course of obstruction. The smooth muscle/collagen ratio increased significantly during the course of obstruction. The expression of ROKalpha increased significantly to approximately the same extent in 1-4-week obstructed groups and increased further in the 8-week obstructed group, both at the mRNA and protein levels. In contrast, there was a significant decrease in the expression of ROKbeta in the obstructed groups, which gradually decrease during the course of 1-4-week obstruction period and are slightly upregulated at the decompensated stage at 8-week obstruction. CONCLUSIONS: The change in the isoforms of ROK may be part of the molecular mechanism for bladder compensation following partial bladder outlet obstruction.

The effect of maturation and age on oestrogen-induced functional hypertrophy of the female rabbit bladder.

OBJECTIVE: To evaluate the effect of maturation and ageing on oestrogen-induced functional hypertrophy of the female rabbit bladder. MATERIALS AND METHODS: Twenty female rabbits were separated into two groups of 10 each by age, young (immature) and old rabbits and each age group was subdivided into three subgroups. The rabbits in subgroup 1 were controls, subgroup 2 were ovariectomized (Ovx) and subgroup 3 were Ovx and received 17-beta oestradiol (1 mg/kg/day) by a subcutaneous slow-release tablet implant. After 15 days of treatment, the rabbits were killed, the bladder was excised, and the body and base separated; two full-thickness longitudinal strips from the ventral surface of the bladder body, and one full-thickness strip from the base, were prepared for contractile studies. The contractile responses to electrical-field stimulation, carbachol, ATP and KCl were determined for both the bladder body and base strips. In addition, full-thickness strips of bladder body and base were fixed in formalin for histological and immunohistological studies. RESULTS: Ovx plus oestradiol resulted in significant increases in bladder weight and responses to all forms of stimulation in young and old rabbits (except for the response to KCl). Vascular density and the smooth muscle (SM)/collagen ratio significantly increased after oestradiol replacement. Interestingly, the increase in vascular density was greater in the young than in the old rabbits. CONCLUSIONS: The present study shows that oestrogen supplementation mediates a functional hypertrophy characterized by increased contractile responses to all forms of stimulation in both young and old rabbits. The increased contractile responses might be explained by the increases in vascular density and SM/collagen ratio.

The effects of cyclical oestrogen on bladder and urethral structure and function.

OBJECTIVE: To determine the effects of cycling oestrogen in rabbits, as oestrogen is essential for physiological maintenance and integrity of the female urogenital tract. MATERIALS AND METHODS: Changes in circulating oestrogen have marked effects on the bladder of experimental animals, with ovariectomy (Ovx) inducing smooth muscle (SM) and mucosal atrophy, increasing collagen synthesis and deposition, decreasing contractile function, mucosal and SM blood flow; oestrogen reverses these effects and increases bladder mass and SM density, primarily by stimulating angiogenesis and increasing blood flow. Twenty adult female New Zealand White rabbits were divided into five equal groups; group 1 served as the control group, and groups 2-5 had a bilateral Ovx. Group 2 received no oestradiol and was assessed 2 weeks after Ovx; groups 3-5 received 17-beta oestradiol from a subcutaneous slow-release tablet 2 weeks after Ovx, which remained in place for a subsequent 2-week period. Group 3 was then assessed after the 2 weeks on oestradiol. Groups 4 and 5 then had their oestradiol tablets removed for 2 weeks and group 4 was assessed after this period off oestradiol. Group 5 then received a new oestradiol tablet that was left in place for an additional 2 weeks. RESULTS: Both groups receiving oestrogen (3 and 5) had a statistically significantly greater bladder weight than both the control group and group 2. The volume fraction of SM paralleled the bladder weight, showing that oestrogen increased the volume fraction of SM whereas Ovx and low oestrogen decreased the SM fraction. The cross-sections of the urethra from groups 3 and 5 were significantly wider than those of either the control or group 1, also being consistent with the structural effects of oestrogen. Intra-arterial phenylephrine increased urethral pressure to a similar level in all groups. The urethral pressure response to intra-arterial acetylcholine shifted from contraction in the control to relaxation in the oestrogen-treated groups. Ovx resulted in a lower vascular density, whereas oestrogen resulted in a significant increase in vascular density (angiogenesis). CONCLUSIONS: Cyclical oestrogen had pronounced structural and pharmacological effects. Low oestrogen decreased the volume fraction of SM, increased collagen, and decreased vasculature, whereas oestrogen mediated a marked hypertrophy of the SM components, decreased the collagen component, and stimulated angiogenesis. Cyclical oestrogen also had marked effects on the responses to intra-arterial acetylcholine, shifting the response from contraction to relaxation.

Novel biomarkers of bladder decompensation after partial bladder obstruction.

AIMS: Partial bladder outlet obstruction (PBOO) results in marked contractile, biochemical, and histological alterations in the bladder. Our aim was to determine the time course of progressive PBOO in the rabbit and to find parameters that marked the shift to decompensation. MATERIALS AND METHODS: Twenty-four rabbits were subjected to 1, 2, 4, and 8 weeks of PBOO. Sham operated rabbits served as controls. At each time period, cystometry was performed and individual bladder strips were used for contractility studies. Full-thickness sections of bladder body from each rabbit were fixed in formalin and used to determine the vascular density and nerve density. The balance of the bladder body was separated between muscle and mucosa and was analyzed for superoxide dismutase (SOD) and catalase (CAT) activities. RESULTS: Bladder weight increased progressively and all contractile responses were reduced significantly over the course of obstruction. Markedly increased bladder weight and very large bladder volumes indicated decompensation. Nerve density was marked decreased in decompensated bladders. Similarly, SOD activity in muscle decreased progressively and was markedly lower in decompensated bladders. Although CAT activity of the muscle increased after 2-4 weeks of obstruction, it decreased markedly in decompensated bladders. CONCLUSION: This study shows that prolonged PBOO causes progressive deterioration in the rabbit bladder with decompensation after 8 weeks. Markedly decreased nerve density and severely reduced SOD and CAT activities are associated with the shift from compensated to decompensated function of the bladder. They may be excellent biomarkers of decompensation.

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit.

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.

Estrogen induced functional hypertrophy and increased force generation of the female rabbit bladder.

AIMS: Estrogen is essential for physiological maintenance of the female urogenital tract. It is believed that alterations in female sex hormones play a major role in the etiology and response to urinary tract dysfunctions. In animal studies, ovariectomy (Ovx) results in smooth muscle (SM) weakness and atrophy whereas estrogen supplementation reverses these effects. Our study seeks to establish the mechanisms by which estrogen augmentation results in increased contractility. METHODS: Twenty New Zealand White female rabbits were separated into five groups of four each. Group 1 served as control, rabbits of groups 2-5 were ovariectomized, group 2 ovariectomized received no estradiol, groups 3-5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous slow release tablet implant for 1, 3, and 7 days, respectively, beginning 2 weeks after Ovx. At the end of the experimental period, each rabbit was anesthetized and the urinary bladder was removed for contractile, histological, and biochemical studies. RESULTS: Ovx resulted in significantly decreased bladder contractile function, whereas bladders tested after estradiol administration showed increased contractility. Ovx resulted in a decrease in SM/collagen ratio, whereas estrogen resulted in an increase. The estrogen receptor (ER) density significantly increased following Ovx. After 1 day of estrogen treatment, the ER density decreased significantly below control levels, but rose progressively during the estrogen treatment. CONCLUSION: The present study demonstrates that estrogen supplementation mediates a "functional hypertrophy," that is a hypertrophy characterized by increased contractile responses to all forms of stimulation, and an increased ratio of SM/collagen.

The immediate effect of castration on female rabbit bladder blood flow and tissue oxygenation.

BACKGROUND: The female urinary bladder is a target organ for estrogen. Reductions in circulating estrogen have been associated with urothelial and vaginal atrophy and bladder disorders including incontinence and increased incidence of bladder infections. We determined the effect of short-term ovariectomy on sex hormones, bladder blood flow, and tissue oxygenation in the rabbit model. MATERIALS AND METHODS: Female New Zealand White rabbits were ovariectomized and evaluated on 1, 3, and 7 days after ovariectomy. Tissue oxygenation (pO2) and blood flow were measured with oxylab system of real time measurements. Serum estrogen and progesterone were determined at sacrifice. Tissue hypoxia was localized histologically using Hypoxyprobe-1 immunohistochemistry. RESULTS: Short-term ovariectomy caused rapid decreases in serum estrogen and progesterone, significant decreases in urothelial oxygenation and blood flow. No significant decreases in blood flow or oxygenation were noted for the detrusor smooth muscle. Immunohistochemistry confirmed the presence of urothelial hypoxia at all times after ovariectomy. Bladder muscle did not demonstrate significant levels of hypoxia. CONCLUSION: The bladder urothelium is extremely sensitive to short-term ovariectomy, with significant urothelial hypoxia seen by post-ovariectomy day 1. Urothelial hypoxia may play a significant role in pelvic pain syndromes, incontinence, and increased susceptibility to bladder infection.

Effect of nitric oxide synthase inhibition on changes induced by estradiol in bladders from ovariectomized rabbits.

PURPOSE: Our current study was designed to determine whether estradiol-induced increases in bladder blood flow could be inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME), and thus whether nitric oxide was involved in estrogen-linked female bladder blood flow alterations. MATERIALS AND METHODS: Sixteen female New Zealand White rabbits were separated into 4 groups of 4 rabbits each. (1) Sham group received sham operation and injections of vehicle (peanut oil). (2) Ovariectomy (OVX) group received ovariectomies and injections of vehicle. (3) Ovariectomy+estrogen (OVX+E) group received ovariectomy and injections of 17beta-estradiol (1 mg/kg) dissolved in peanut oil. (4) Ovariectomy+estradiol+L-NAME (OVX+E+L-NAME) group received ovariectomies and injections of 17beta-estradiol and L-NAME. All treatments were continued for 4 weeks. At 4 weeks after treatment, each rabbit was anesthetized and cystometries were performed. After cystometry, blood flow to the detrusor muscle and mucosa was determined by standard fluorescent microsphere infusion technique. Then four longitudinal detrusor strips and two rings of descending thoracic aorta were mounted in individual 15 ml baths containing oxygenated Tyrode's solution at 37 degrees C. Contractile responses to several agents were determined. Full-thickness sections of detrusor were fixed and embedded in paraffin for alpha-actin immunostaining. RESULTS: In the bladder: (1) Estradiol resulted in an increases in bladder weight and blood flow; L-NAME inhibited these increases. (2) OVX resulted in a decreased cystometric capacity; estradiol resulted in increased capacity which was attenuated by L-NAME treatment. (3) OVX resulted in significantly decreased contractile responses to all forms of stimulation; estradiol resulted in significantly increased contractile responses which were attenuated by L-NAME treatment. (4) OVX resulted in a significant decrease in the volume-fraction of smooth muscle in the detrusor; estradiol resulted in a significant increase which was attenuated by L-NAME. CONCLUSIONS: These findings strongly support the hypothesis that nitric oxide plays a major role in the alterations in blood flow mediated by changing circulating estrogen and that these changes mediate at least in part the cystometric and contractile changes induced by alterations in circulating estrogen.

Effect of ethanol on protection of urinary bladder function by grape suspensions.

OBJECTIVES: To compare the protective effects of grape suspensions prepared in an aqueous vehicle with grape suspensions prepared in an 8% ethanol vehicle in rabbits subjected to partial outlet obstruction. The hypothesis was that the presence of ethanol would increase the absorption of the beneficial components of the grape suspensions and thus increase their protective ability. The use of ethanol in these studies was not to simulate wine. METHODS: A total of 48 New Zealand white rabbits were separated into eight groups of 6 rabbits each. Groups 1 and 3 were pretreated by oral gavage for 3 weeks with grape suspensions in water; groups 2 and 4 were treated with vehicle. Groups 5 and 7 were treated with the grape suspensions in 8% ethanol, and groups 6 and 8 were treated with ethanol vehicle. Groups 1, 2, 5, and 6 underwent sham operations, and groups 3, 4, 7, and 8 underwent partial outlet obstruction. Three weeks after surgery, the rabbits were evaluated. RESULTS: The bladder weight had significantly increased in all obstructed groups. The contractile responses to field stimulation and carbachol were reduced in all obstructed groups, although the responses in both grape-treated groups were greater than both vehicle-treated groups. The contractile responses to potassium chloride were significantly reduced by partial outlet obstruction in both obstructed groups similarly. CONCLUSIONS: Both grape suspensions provided protection against obstructive-induced bladder dysfunction. The ethanol preparation of the grape suspension was not better than the aqueous preparation.

Effect of age on rabbit bladder function and structure following partial outlet obstruction.

PURPOSE: We determined whether young and old rabbits respond differently to partial bladder outlet obstruction. MATERIALS AND METHODS: A total of 16 male New Zealand White rabbits were separated into 2 groups of 8 each. Group 1 consisted of young rabbits (age 7 weeks) and group 2 consisted of old rabbits (age 2 years). Four rabbits per group underwent partial outlet obstructions and 4 underwent sham operation. Four weeks following surgery individual bladder strips were used for contractile studies and the remaining tissue was examined histologically. RESULTS: Contractile responses to all forms of stimulation between the young and old sham operated groups were similar. Contractile responses to all forms of stimulation were significantly decreased to the same degree in the 2 obstructed groups. However, the rate of tension generation to field stimulation was decreased to a significantly greater degree in young vs old bladders. Although young and old bladders showed smooth muscle hypertrophy, older rabbits showed significantly greater thickening of the serosa than young rabbits. Young rabbits showed significant inflammation, hemorrhage and expansion of the lamina propria, whereas old rabbits showed none of these characteristics. CONCLUSIONS: Although there were only minor differences in the physiological response of young and old bladders to obstruction, young rabbits showed a significantly greater degree of histological damage. This may have been due to the thinner wall and greater sensitivity to distention.