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Obstructive bladder dysfunction is directly related to ischemia/reperfusion injury characterized by damage to nerves, synapses and smooth muscle cells within the bladder wall. Antrodia Camphorata (AC) has significant antioxidant, antiinflammatory and cell-cycle inhibition properties. The specific aim of this study was to evaluate whether orally administered AC can protect rabbit bladders from the progressive dysfunctions induced by bilateral ischemia/reperfusion (I/R).Twenty-four male NZW rabbits were separated into 4 groups of 6 animals each. Rabbits in groups 1 and 2 were fed Antrodia Camphorata (AC) suspensions; those in groups 3 and 4 received vehicle. Each rabbit in groups 2 and 4 were subjected to in vivo bilateral ischemia for 2 h and then allowed to recover for 1 week. The rabbits in groups 1 and 3 received sham operation and served as control groups. Cystometry, contractile responses to field stimulation, carbachol, ATP and KCl were determined. Biochemical and immuno-histochemical studies were also performed.I/R resulted in decreased compliance, decreased contractile responses, decreased nerve density, and increased apoptosis. AC pretreatment of rabbits subjected to I/R significantly protected the bladder from all contractile, biochemical, and structural dysfunctions resulting in significantly improved bladder.

Objectives To detect the effect of ischemia/reperfusion (I/R) injury on rabbit bladder, using physiological study and immunoblotting techniques. Methods Twelve male New Zealand White rabbits were separated into three groups of 4 rabbits each. Group 1 served as control. Group 2 rabbits (ischemia-alone group) underwent in vitro bilateral ischemia surgery for 2 hours. In group 3 (I/R group), bilateral ischemia was similarly induced, and the rabbits were allowed to recover for 2 weeks. The contractile responses to electrical field stimulation, adenosine triphosphate, carbachol, and KCl were recorded. Expression levels of the signaling targets, Rho-kinase (ROK), protein kinase C potentiated inhibitor (CPI-17), caldesmon (CaD), and calponin (CaP) were analyzed by Western blotting. Results Ischemia alone resulted in significant reductions in the contractile responses, whereas I/R resulted in further decreases after all forms of stimulation. In muscle layer, ROK expression increased immediately after ischemia and recovered to the control level after 2 weeks' recovery. However, in mucosa layer, ROK expression showed no significant change after ischemia but significantly increased after reperfusion. After ischemic damage, CPI-17, the functional protein involved in smooth-muscle Ca 2+ sensitization, was significantly increased and then decreased after 2 weeks of reperfusion. The expression of CaP significantly increased after ischemia and decreased after reperfusion. Levels of high-molecular-weight CaD significantly decreased after ischemia and remained very low after reperfusion. Conclusions This study provides further understanding of the role of regulatory proteins in detrusor muscle after ischemia and I/R-induced contractile dysfunction.

Obstructive bladder dysfunction is in part due to reduced blood flow and the resulting ischemia of the bladder smooth muscle and mucosa. Our aim was to determine if the severity and localization of ischemia could be determined by measuring blood flow to the bladder with a non-invasive probe placed on the surface of the urothelium. Twenty-four adult male rabbits (5 months, 3.5–4.0 kg) were divided into three groups: 1—controls; 2—2 h of bilateral ischemia; and 3—partial outlet obstruction, and were evaluated after 2 weeks. Each rabbit received an intraperitoneal injection of Hypoxyprobe-1. In vivo real-time monitoring of blood flow was measured at five sites within the bladders with a laser Doppler flowmeter. For all groups, the blood flow readings showed no significant differences among the five sites. The ischemic bladders showed significant decreases in blood flow. The obstructed bladders had significantly lower blood flow than the ischemic bladders. The hypoxyprobe studies demonstrated that there was no hypoxia present in the control bladders; the mucosa of the ischemic bladders showed even hypoxia at an intermediate concentration; the obstructed bladders showed dense but even staining. We have demonstrated that we can determine the severity of ischemia by surface measurement of blood flow.

Recent evidence indicates that ischemia and reperfusion are major etiological factors in the bladder dysfunction that occurs after partial bladder outlet obstruction. Coenzyme Q10 and alpha-lipoic acid are found naturally in mitochondria and act as potent antioxidants. We investigated the beneficial effects of coenzyme Q10 plus alpha-lipoic acid in a rabbit model of bladder outlet obstruction.Twenty male rabbits were divided into 5 groups. Group 1 served as control and group 2 received three weeks of coenzyme Q10 plus alpha-lipoic acid supplementation. Rabbits in group 3 underwent surgical partial bladder outlet obstruction for duration of four weeks and groups 4 and 5 were obstructed for seven weeks. In group 5, coenzyme Q10 plus alpha-lipoic acid supplementation was given following 4 weeks obstruction and continued till the end of the seven weeks. The contractile responses to various agents were determined. The protein nitration and carbonylation levels were studied by immunoblotting. Nerve function was determined by choline acetyltransferase activity and nerve density.The contractile responses to different forms of stimulations, including field stimulation, ATP, carbachol and KCl all showed decreases following 4 and 7 weeks obstruction. Treatment with coenzyme Q10 plus alpha-lipoic acid significantly restored contractile responses to all forms of stimulation. Treatment also had mitochondrial and neuronal effects and reduced protein nitration and carbonylation. Histologically there was less detrusor muscle hypertrophy.The current study clearly demonstrates that coenzyme Q10 and alpha-lipoic acid supplementation can improve bladder function after outlet obstruction.

OBJECTIVE To measure the degree to which partial bladder outlet obstruction (PBOO) results in oxidative bladder damage, which subcellular components of the bladder are affected and whether these changes correlate with bladder function. MATERIALS AND METHODS In all, 32 rabbits were divided into four groups. Each group underwent PBOO for 1, 2, 4, and 8 weeks, respectively. Bladder tissue from each group was homogenized and separated into subcellular fractions via differential centrifugation. The carbonyl content within the subcellular fractions, including the nuclear, mitochondrial, and microsomal pellets, was then quantified by dot blot analysis. RESULTS Total bladder oxidation increased with duration of obstruction across all subcellular fractions. The largest increase in total oxidation occurred between 4 and 8 weeks. Protein oxidation density in the nuclear and microsomal fractions both showed increases at 2 weeks obstruction, decreases at 4 weeks, and then large increases at 8 weeks. The increase in protein oxidation density between 4 and 8 weeks obstruction was most pronounced in the microsomal fraction. CONCLUSIONS Overall bladder protein oxidation increased with the duration of obstruction and increased at a greater rate during the transition to decompensation. Furthermore, the subcellular fraction that exhibited the most oxidation was the microsomal pellet. The amount of protein oxidation correlated with the functional changes in the 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.

Incontinence in women is a major health problem. In a recent study, incontinence affects more than 10 million Americans, 85% of whom are women. Incontinence can result in an extremely altered life style, and is the main reason that women are admitted to nursing homes. Care for the incontinent patient represents an enormous expense nationally. The emphasis of this review is on the relevance of estrogen-dependent ischemia to urethral smooth muscle tone and tonic � -adrenergic tension in the maintenance of continence in women. Additionally, the role of ischemia in bladder dysfunc- tion will be explored. In general, the relative contribution of smooth muscle tension is greater in females than in males. It is clear from both animal and human data that during bladder filling there is a progressive increase in sympathetic stimula- tion of the urethra resulting in increased � -adrenergic tension.

Purpose Ischemia, reperfusion, and free radical generation have been recently implicated in the progressive bladder dysfunction. Coenzyme Q10 (CoQ10) is a pro-vitamin like substance that appears to be efficient for treatment of neurodegenerative disorders and ischemic heart disease. Our goal was to investigate the potential protective effect of CoQ10 in a rabbit model of in vivo bilateral ischemia and ischemia/reperfusion (I/R). Material and Methods Six groups of four male New Zealand White rabbits each were treated with CoQ10 (3 mg/kg body weight/day—dissolved in peanut oil) (groups 1–3) or vehicle (peanut oil) (groups 4–6). Groups 1 and 4 (ischemia-alone groups) had clamped bilateral vesical arteries for 2 h; in groups 2 and 5 (I/R groups), bilateral ischemia was similarly induced and the rabbits were allowed to recover for 2 weeks. Groups 3 and 6 were controls (shams) and were exposed to sham surgery. The effects on contractile responses to various stimulations and biochemical studies such as citrate synthase (CS), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and catalase (CAT) were evaluated. The protein peroxidation indicator, carbonyl group, and nitrotyrosine contents were analyzed by Western blotting. Results Ischemia resulted in significant reductions in the contractile responses to all forms of stimulation in vehicle-fed rabbits, whereas there were no reductions in CoQ10-treated rabbits. Contractile responses were significantly reduced in vehicle-treated I/R groups, but significantly improved in CoQ10-treated rabbits. Protein carbonylation and nitration increased significantly in ischemia-alone and I/R bladders; CoQ10 treatment significantly attenuated protein carbonylation and nitration. CoQ10 up-regulated SOD and CAT activities in control animals; the few differences in CoQ10-treated animal in SOD and CAT after ischemia and in general increase CAT activities following I/R. Conclusions CoQ10 supplementation provides bladder protection against I/R injury. This protection effect improves mitochondrial function during I/R by repleting mitochondrial CoQ10 stores and potentiating their antioxidant properties.

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.

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.

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca2+ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.

Previous studies have demonstrated that ovariectomy induces reduced blood flow and hypoxia, resulting in free radical damage of the mucosal and smooth muscle compartments of the rabbit urinary bladder, whereas estradiol administration results in angiogenesis and recovery from hypoxia. The current study was designed to investigate the effects of ovariectomy and estradiol replacement on the superoxide dismutase (SOD) and catalase (CAT) activities of the bladder.A total of 12 mature female rabbits were divided into three groups of four rabbits each: control, ovariectomy, and ovariectomy with 17-beta estradiol supplementation by subcutaneous slow-release tablet. The bladder body and base of the rabbits were examined after 2 weeks. The bladder body and base were separated into muscle and mucosa, and the tissues were analyzed for SOD and CAT activities.Quantitative SOD activities for the mucosa and muscle of both bladder body and base increased after ovariectomy when compared with those of controls. Estradiol replacement resulted in a significant decrease in the SOD activities in the body muscle. Ovariectomy caused a decrease in the CAT activities in the bladder tissues, whereas estradiol treatment resulted in significant increases.These data indicate that ovariectomy induced generation of reactive oxygen species (ROS), as evidenced by the enhanced SOD activity, indicating oxidative stress in the lower urinary tract. Estradiol replacement reversed the effects of ovariectomy; this finding suggests an anti-oxidant effect of estradiol on the bladder.

Objectives Estrogen is essential to mediate physiologic functions in female bladders. Deficiency of estrogen has been speculated to be an etiologic factor for bladder dysfunction in postmenopausal women. Our previous studies have demonstrated that estrogen supplementation in female rabbits induces a “functional hypertrophy” of the urinary bladder smooth muscle. The present study investigated the alterations in the contractile and regulatory proteins in this model. Methods Twenty New Zealand white female rabbits were separated into five groups of 4 rabbits each. Group 1 served as the control, groups 2 to 6 underwent ovariectomy (Ovx), and group 2 served as the Ovx without estradiol treatment group. Two weeks after Ovx, groups 3 to 5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous implant for 1, 3, and 7 days, respectively. The expression of the contractile and regulatory proteins, such as myosin light chain kinase, rho-kinase, and caldesmon, was analyzed by Western blotting. Results The expression of myosin light chain kinase was enhanced by estradiol supplementation. The expression of rho-kinase-alpha was increased significantly (20-fold) after Ovx, which was downregulated after estrogen supplementation. No significant change was seen in rho-kinase-beta after Ovx or estradiol supplementation. The expression of caldesmon isoforms was enhanced by 1-day estradiol supplementation but decreased to lower levels than those of the control group by 3 and 7 days of estrogen treatment. Conclusions The results of the present study have provided more understanding about the role of the contractile and regulatory proteins in detrusor muscle, in both dysfunctional atrophy induced by Ovx and functional hypertrophy induced by estrogen supplementation.

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-β 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.

Partial bladder outlet obstruction (PBOO) results in cellular damage due to ischemia and reperfusion injury. Our study seeks to establish how early this damage can occur and the role that nitric oxide may play in its pathophysiology. Surgical PBOO (1, 3, and 7 days) were performed on male New Zealand White rabbits. Half of the animals were premedicated for 3 days with NG-nitro-l-arginine methyl ester(l-NAME), an inhibitor of nitric oxide synthase before obstruction. Bladder weight increased with duration of PBOO but was significantly lower at 3 and 7 days in animals treated with l-NAME compared with their untreated counterparts. Contractile function decreased progressively with PBOO duration. At 1 day postobstruction, bladder contractility was significantly lower in the l-NAME rabbits than in the untreated rabbits. At 3 and 7 days, contractility of the l-NAME bladders was equal or higher than the untreated bladders. The level of hypoxia at 1 day after obstruction was significantly higher in the l-NAME-treated animals than in the untreated controls but equal at 3 and 7 days obstruction. Increased nitrotyrosine was seen by Western blot in all obstructed animals. However, the amount was significantly less in the l-NAME-treated animals at 3 and especially at 7 days. Nerve density decreased progressively after obstruction; however, it decreased to a significantly lesser degree in the l-NAME-treated bladders than in the untreated groups. These results suggest that l-NAME pretreatment enhanced ischemic damage at 1 day after obstruction but protected the bladder from nitric oxide-generated free radical damage at the later time periods by inhibiting the generation of nitrotyrosine.

Aims It has been demonstrated that partial bladder outlet obstruction (PBOO) causes free radical generation that, in turn, results in cellular and subcellular damage. We tested the hypothesis that nitration of proteins is associated with contractile dysfunctions in obstructive bladder disease. Methods Thirty rabbits were subjected to 1–28 days of partial outlet obstruction. Sham operated rabbits served as controls. Western blotting was used to determine the amount of nitrotyrosine level at the protein level. At each time point, isolated strips of bladder body were mounted in individual baths and the contractile response to field stimulation (FS), carbachol, and KCl determined. Results Bladder weight increased rapidly during the first 7 days and then increased slowly thereafter. There was a fourfold increase in the amount of nitrotyrosine in the 7 day obstructed groups when compared to sham controls and the levels remain elevated at 14 and 28 days of obstruction. Contractile dysfunction in response to FS (8 and 32 Hz) was noted as early as 1 day after obstruction and increased progressively over the study period. The decrease in response to carbachol and KCl was significant only after 3 days of obstruction and the progressive increase in dysfunction was slower than with FS. Conclusions PBOO is accompanied by an increase in nitrotyrosine, a marker of free radical damage. Simultaneously there was a progressive decrease in contractility of detrusor smooth muscles (DSMs). Nitrotyrosine may be usable as a marker of free radical damage and reperfusion injury. Neurourol. Urodynam. © 2006 Wiley-Liss, Inc.

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-β 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. Neurourol. Urodynam. © 2006 Wiley-Liss, Inc.

OBJECTIVE To investigate the potential protective effect of a grape suspension in a rabbit model of in vivo bilateral ischaemia/reperfusion (I/R), which is a causal factor in obstructive bladder dysfunction. MATERIALS AND METHODS Six groups of four New Zealand White rabbits were treated by twice-daily gavage with aqueous grape suspension (groups 1–3) or sugar-water vehicle (groups 4–6) for 3 weeks. Groups 1 and 4 then received bilateral ischaemia for 2 h, and groups 2 and 5 received bilateral ischaemia for 2 h and reperfusion (recovery) for 1 week. Groups 3 and 6 were controls (sham-operated). The effects on cystometry, in vitro contractile responses, and morphology were evaluated. RESULTS Ischaemia resulted in significant reductions in the contractile responses to all forms of stimulation in vehicle-fed rabbits, whereas there were no reductions in grape-fed rabbits. Contractile responses were significantly reduced in both I/R groups, but significantly more in vehicle-fed than in grape-fed rabbits. Immunohistochemical studies showed less hypoxia in the bladders of grape-fed rabbits than in vehicle-fed rabbits for both ischaemia-only and I/R groups. CONCLUSIONS Feeding rabbits with grape suspension provided significant protection against the hypoxic effects of bilateral ischaemia and I/R.

Purpose: The thin filament associated proteins caldesmon, tropomyosin and calponin have been shown to modulate actin-myosin interaction, actomyosin adenosine triphosphatase and contraction in smooth muscle. This study was performed to determine whether the expression of these proteins is altered in diabetes induced decrease in the contractility of bladder wall smooth muscle. Materials and Methods: Detrusor samples were obtained from New Zealand White male rabbits with alloxan induced diabetes, and from age and sex matched control rabbits. In addition, a bladder myocyte cell line, which continues to express smooth muscle phenotype, was exposed to either normal (5 mM) or high (50 mM) concentrations of glucose. The levels of expression of the thin filament associated proteins were determined at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Results: Detrusor smooth muscle tissue from rabbits with alloxan induced diabetes showed over expression of thin filament associated proteins, calponin, tropomyosin and caldesmon when compared with that of the control. Similar up-regulation was seen also in bladder myocytes in cultures treated with 50 mM glucose, indicating that the high glucose induced the changes. Conclusions: Our results suggest that the increased expression of thin filament proteins, calponin, tropomyosin and caldesmon in diabetic rabbits might alter the contractile and cytoskeletal structure in bladder myocytes. The over expression of these thin filament associated proteins, which suppresses actin-myosin interaction and actomyosin adenosine triphosphatase, and the enhancement of this suppression by tropomyosin are likely to have an effect on the relationship between force and myosin light chain phosphorylation, requiring higher levels of phosphorylation in diabetic detrusor compared with that of control. The downstream effects of high glucose (eg oxidative stress) appear to modulate the transcriptional regulation of thin filament mediated regulatory proteins in bladder smooth muscle.

Aims Obstruction of the urinary bladder outlet induces detrusor smooth muscle (DSM) hypertrophy. The goal of this study was to determine whether the composition of thin filament-associated proteins, known to play important roles in cytoskeletal structure and/or the regulation of contraction, is altered in DSM during hypertrophy. Methods DSM hypertrophy was induced in male rabbits by partial ligation of the urethra. Sham-operated rabbits served as a control. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR revealed a significant increase in the expression of mRNAs for basic (h1) calponin (CaP), and α-isoform of tropomyosin (Tm) in hypertrophied DSM compared to controls. Western blotting and two-dimensional (2-D) gel electrophoresis showed enhanced expression of these proteins and also a significant increase in the expression of β-non muscle and γ-smooth muscle actin in the DSM from obstructed bladders, while α-actin remained constant. Results Enhanced expression of these proteins in the DSM from obstructed bladders was confirmed by immunofluorescence microscopy. Double immunostaining with Cap/Tm and α/β-actin-specific antibodies showed co-localization of these proteins in myocytes. Colocalization of smooth muscle specific myosin and CaP to cytoplasmic filaments in cells dissociated from the hypertrophied DSM indicated that these cells are differentiated smooth muscle cells. Conclusions The change in the isoforms of actin, Cap, and Tm may be part of the molecular mechanism for bladder compensation in increased urethral resistance. Neurourol. Urodynam. © 2005 Wiley-Liss, Inc.

Objectives To evaluate the effect ovariectomy (OVX) after 2 and 4 weeks on bladder function and biochemistry of the adult female rabbit urinary bladder. Methods Twelve mature female rabbits were divided into 3 groups: control, 2-week ovariectomized, and 4-week ovariectomized. At the end of the experimental period, the following studies were performed: contractile studies on isolated strips; examinations of the activity of citrate synthase (a marker for mitochondrial function) and thapsigargin-sensitive calcium ATPase (a marker for sarcoplasmic reticular calcium uptake function); and quantification of Rho-kinase (ROK) α and β and myosin light chain kinase by Western blot analyses. Results By 28 days after OVX, there were significant decreases in bladder weight, contractile responses, and citrate synthase and sarcoplasmic reticular calcium uptake activity. In addition, by 28 days following OVX the relative concentration of ROK α was significantly increased, whereas ROK β concentration was significantly decreased. Myosin light chain kinase was significantly reduced. Conclusions Our study demonstrated that OVX contributed significantly to chronically decreased contractile function in the detrusor muscle of the female rabbit bladder, and this decrease, in turn, was mediated by decreased mitochondrial and sarcoplasmic reticulum function. These specific bladder dysfunctions could be related to the demonstrated decreased blood flow to the bladder muscle and mucosa and the increased generation of free radicals. Changes in smooth muscle regulatory proteins, especially myosin light chain kinase, may also play a role in contractile dysfunctions.

Aims Ischemia/reperfusion (I/R) can significantly change the nerve function of the bladder, thus resulting in detrusor weakness and overactivity. CoQ10 is a lipid-soluble cofactor found naturally in the mitochondria and has been reported to have neuroprotective and antiapoptosis effects. The aim of this study is to determine if CoQ10 can protect bladders subjected to I/R injury. Methods Four groups of male New Zealand White rabbits (N = 4) were treated with CoQ10 (3 mg/kg body weight/day) (groups 1and 2) or vehicle (groups 3 and 4). In groups 2 and 4 (I/R groups), bilateral vesicular ischemia was induced for 2 hr and the rabbits allowed to recover for 2 weeks. Groups 1 and 3 were controls and given sham surgery. The cholinergic nerve marker, vesicular acetylcholine transporter (VAChT), was examined by western blotting. Nerve density and cell apoptosis were calculated by immunohistochemistry. Results I/R significantly decrease bladder innervation; CoQ10 has significant neuroprotective effects, which are evidenced by increased VAChT expression and neurofilament immunostaining. Detrusor cells apoptosis increase significantly by I/R. CoQ10 control and I/R groups both show significantly lower apoptosis than vehicle groups. Conclusions The current study clearly demonstrated that these CoQ10 supplement provides significant bladder protection against I/R injury. This protective effect is in part by protecting damage to cholinergic innervation. Neurourol. Urodynam. 28:339–342, 2009. © 2008 Wiley-Liss, Inc.

Aims Estrogen administration to female rabbits induces a functional hypertrophy of the bladder. The aim of this study was to investigate whether supplementation of estrogen in the female rabbit with partial bladder outlet obstruction (PBOO) would affect the severity of bladder dysfunction. Methods We surgically created PBOO in female New Zealand White rabbits. Group 1 included sham operated rabbits which served as controls. Group 2 received PBOO without estrogen treatment. Group 3 received estrogen treatment after PBOO. Group 4 received estrogen pretreatment before PBOO. The bladders were then removed for contractile, biochemical, and protein expression studies. There were four rabbits per group. Results (1) PBOO resulted in significant decreases in the contractile responses to all forms of stimulation (field stimulation [FS], carbachol, KCl, ATP). Both pretreatment and post-treatment with estrogen resulted in significantly increased contractile responses to all forms of stimulation, although the responses were still lower than control. (2) PBOO resulted in a significant decrease in the activity of choline acetyltransferase (ChAT). Both pretreatment and post-treatment with estrogen resulted in significant increases in ChAT activity back toward control levels. (3) PBOO resulted in significant increases in both protein oxidation and nitration; both pretreatment and post-treatment with estrogen significantly reduced oxidation and nitration toward control levels. Conclusions Estrogen pretreatment and post-treatment in the female rabbit ameliorated contractile and biochemical dysfunctions associated with PBOO. This improvement is likely due to reduced oxidative stress. As expected, pretreatment was generally more effective than post-treatment. Neurourol. Urodynam. 28:95–100, 2009. © 2008 Wiley-Liss, Inc.

Objectives To investigate the effects of inosine on in vitro ischemia-reperfusion injury to urinary bladders in adult rats. Inosine has neuroprotective effects against cerebral and cardiac ischemia-reperfusion injury. Methods A total of 18 adult male rats were used. Each rat was anesthetized, and the bladder was excised, cut longitudinally into 3 equal strips, and mounted in individual baths. Six strips were run simultaneously. Inosine (37.3 mM) was added to the baths containing strips 2, 4, and 6. All strips were equilibrated for 1 hour in oxygenated Tyrode solution containing glucose and at the end of the hour were stimulated with field stimulation, carbachol, and KCl. Strips 1 and 2 were subjected to repetitive stimulation (RS) by field stimulation (32 Hz) every 5 minutes for 1 hour; strips 3 and 4 were subjected to Tyrode equilibrated solution with nitrogen and no glucose for 1 hour; and strips 5 and 6 were subjected to in vitro hypoxia in the absence of substrate plus RS for 1 hour. All strips were then incubated in oxygenated Tyrode solution for 1 hour and stimulated as before. Results In normoxia, inosine had no beneficial effects either before or after RS. However, in the ischemic model, the bladder strips incubated with inosine showed significantly better contractile responses to stimulation with carbachol and KCl. After hypoxia without substrate plus RS, the strips incubated with inosine showed increased responses to all 3 forms of stimulation. Conclusions Inosine protected the contractile responses of the bladder strips after in vitro hypoxia without substrate with or without RS.

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. Neurourol. Urodynam. 27:826–831, 2008, © 2008 Wiley-Liss, Inc.

Ischemia, reperfusion, and subsequent free radical damage have been implicated in many voiding disorders. Our goal was to investigate further the mechanisms of these disorders, with particular emphasis on nerve and mitochondrial function and on detrusor smooth-muscle cells. The effects on contractile responses to various stimulations, citrate synthase, choline acetyltransferase activities, and vesicular acetylcholine transporter were evaluated after ischemia alone and ischemia/reperfusion 2 h, 7 days, and 14 days. Nerve density and detrusor cell apoptosis were also measured. The contractile responses were significantly decreased at both 7 and 14 days reperfusion, although at 14 days some recovery was observed. Similar patterns were seen for the intramural nerves, both nerve cell cytoskeletal structures and cholinergic neurotransmitters. Citrate synthase activity was also depressed by ischemia and 2 h reperfusion, but the activity recovered by 7 days. Detrusor cell apoptosis was not significantly affected by ischemia and 2 h reperfusion; but showed an approximately 14-fold increase at both 7 and 14 days reperfusion. Reperfusion following ischemia resulted in worsening intramural bladder nerve dysfunction, nerve fiber injury, mitochondrial injury, and damaged detrusor muscle cells. However, at 14 days reperfusion, nerve and mitochondrial regeneration occurred and resulted in partial recovery of contractile function.

OBJECTIVE To evaluate the effects of ovariectomy (Ovx) and oestrogen therapy on the free fatty acid (FFA) content, endogenous lipase activity, and the phospholipid (PL) content of the urinary bladder, as reduced circulating oestrogen during and after the menopause has been linked to various bladder dysfunctions including incontinence and recurrent urinary tract infections. MATERIALS AND METHODS In all, 12 New Zealand White female rabbits were separated into three groups; the Ovx + oestrogen group received Ovx and treatment with conjugated β-oestradiol (3 weeks), the Ovx group received Ovx and vehicle (3 weeks), and the control group received sham operation and vehicle. Cystometry was used to evaluate compliance and in vitro muscle strip studies quantified contractility. For the bulk of the bladder, the muscle and mucosa were separated; FFA and PL concentrations were analysed using standard biochemical techniques. RESULTS The bladder contractile responses and compliance decreased after Ovx and returned to or above normal after oestrogen administration. Both FFA and PL concentrations of the mucosa were about three times greater than that of the smooth muscle. Ovx significantly reduced the FFA and PL concentrations of both muscle and mucosa, while oestrogen therapy restored them to normal. CONCLUSIONS Reduced FFA and PL content of the smooth muscle membranes would decrease their fluidity and contribute to decreased compliance and contractility. Reduced FFA and PL content of the mucosa would be consistent with mucosal damage and may contribute to the increased incidences of incontinence and bladder infection.

Ovariectomy resulted in decreased blood flow and hypoxia to the bladder mucosa and smooth muscle. Nitric oxide (NO) played an important role in regulating bladder function during bladder ischemia and reperfusion. This study was designed to evaluate the role of NO on bladder function in the first few days after ovariectomy. Female rabbits were separated into three groups, one which received no medication, pre-medicated with N(omega) -nitro-L-arginine methyl ester (L-NAME) and the third treated with L-arginine. Non-ovariectomized controls and at 1 and 3 days post-ovariectomy, animals from each group were euthanized. Cystometry and in vitro isometric contractile responses were recorded and the oxidative stress markers, nitrotyrosine and protein carbonylation were determined. L-NAME treatment did not significantly alter bladder function after ovariectomy. L-arginine fed, ovariectomized rabbits had lower intravesical pressure and better contractile responses to all forms of stimulation than the ovariectomized rabbits with or without L-NAME. Furthermore, the ovariectomized ones with or without L-NAME had higher oxidative stress markers than L-arginine fed rabbits. This study clearly demonstrates that feeding rabbits with L-arginine can protect the bladder from oxidative free radical damage following short term ovariectomy.

Parathyroid hormone-related protein (PTHrP), the main factor responsible for malignant hypercalcemia, is produced by a wide range of normal and malignant tissues. Prior studies in the rabbit model demonstrated that partial bladder outlet obstruction results in calcium-dysregulation characterized by a marked increase in free calcium within the smooth muscle compartment and the stimulation of calcium-activated enzymes, such as calpain and phospholipase A(2).Twenty-four male New Zealand white rabbits were divided into four groups of six each. Following 4 weeks of obstruction, one group of animals was killed, while outlet obstruction was reversed in two additional groups of animals, which were killed 4 and 8 weeks after relieving the obstruction. A group with six sham-operated rabbits served as controls. The expression and localization of PTHrP were detected in muscle and mucosa by immunohistochemistry, using a PTHrP-specific antibody.In the sham-operated group, rabbit bladders showed a low expression of PTHrP in both the mucosa and muscle layers. PTHrP in the 4-week obstructed bladder group, in muscle and mucosa, were 266% and 134% higher than the sham group, respectively. Strong PTHrP immunostaining persisted in the 4-week reversal groups, but it returned to the sham level after 8 weeks of reversal in the muscle layer. As mentioned about the mucosa layer, the PTHrP level returned to control levels more rapidly after 4 weeks of reversal and continued after 8 weeks of reversal.This study showed that PTHrP is increased after partial bladder outlet obstruction and decreased after relieving the obstruction.

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.

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.

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 Ca2+-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.

Objectives We examined the expression of Rho-kinase (ROK) isoforms in young and old rabbits’ detrusor smooth muscles (SM) during the progression of short-term partial bladder outlet obstruction and correlated them with the time course of obstruction. Methods We obtained detrusor samples from bladders after 1, 3, 7, and 14 days of obstruction and also sham-operated control rabbits. We used reverse transcriptase–polymerase chain reaction, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and Western blotting to determine the relative levels of ROK isoform expression at the mRNA and protein levels. Results Bladder weight for young rabbits increased between 1 and 7 days’ obstruction and came back toward control levels at 14 days’ obstruction. In old rabbits, bladder weight increased after obstruction, reaching a maximum at 3 days and remaining at this level throughout the 14 days. In young rabbits, the expression of ROKα increased in 1- to 7-day obstructed groups and decreased in the 14-day group, whereas it increased progressively in the old rabbits at both the mRNA and protein levels. There was a significant decrease in the expression of ROKβ in young obstructed rabbits, which gradually decreased during the course of 1- to 7-day obstruction period and increased after 14 days of obstruction. In old groups, there was a decrease in expression after 1 day of obstruction and values remained at a decreased level throughout the course of the study. Conclusions Young rabbit bladders are better able to adapt to bladder outlet obstruction and ROK isoforms respond in a similar way.

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. Neurourol. Urodynam. 26:1036–1042, 2007. © 2007 Wiley-Liss, Inc.

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 ROKα 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 ROKβ 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. Neurourol. Urodynam. 26:1043–1049, 2007. © 2007 Wiley-Liss, Inc.

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-β 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.

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.

Objectives Recent evidence indicates that ischemia and reperfusion (free radical generation) are major etiologic factors in the rabbit bladder subjected to partial outlet obstruction. The current investigation was designed to determine the time course of damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) by measuring the carbonyl and nitrotyrosine content of the bladder. Methods Male rabbits underwent surgical partial bladder outlet obstruction for durations of 1, 2, 4, and 8 weeks. Sham-operated rabbits served as controls. The contractile responses to field stimulation (32 Hz) and carbachol (20 μmol/L) were determined. The nerve density was determined by using immunohistochemical methods. The carbonyl and nitrotyrosine contents within the bladder body wall and mucosa were analyzed by Western blotting. Results Nerve density showed marked decrease in obstructed bladders. The responses to field stimulation and carbachol showed remarkable decrease in bladders after 8 weeks’ obstruction. The protein carbonylation increased significantly in obstructed bladders when compared with controls, both in mucosa and muscles and reached the highest level in 4 weeks’ obstruction. There was a 2-fold increase for nitrotyrosine in mucosa after 8 weeks of obstruction. The increased expression of nitrotyrosine in muscle was maximized at 4 weeks of obstruction. Conclusions The free radicals mediated protein carbonylation and tyrosine nitration occurs to a significant degree in the partially obstructed rabbit bladder. This may be related with sensory and contractile dysfunction and can be used as markers for free radical damage and reperfusion injury.

Purpose: Ischemia/reperfusion (I/R) is a major etiological factor in the bladder dysfunctions observed in men with lower tract obstruction, women with postmenopausal incontinence and with aging. A standardized grape suspension protects the rabbit urinary bladder from both the contractile dysfunctions and the morphologic changes mediated by I/R. Using a model of in vivo bilateral ischemia/reperfusion, the current study investigated the effect of this grape suspension on the endogenous antioxidant defense systems. Materials and methods: 24 NZW rabbits were separated into 6 groups of 4. Groups 1–3 were treated by gavage with aqueous grape suspensions; groups 4–6 received sugar-water vehicle. Groups 3 and 6 were controls. Groups 1 and 4 were subjected to bilateral ischemia for 2 h (I). Groups 2 and 5 underwent bilateral ischemia for 2 h and reperfusion for 1 week (I/R). For all rabbit bladders, the muscle and mucosa were separated by blunt dissection and analyzed separately. The effects of the various treatments on bladder antioxidant systems of cytoplasmic superoxide dismutase (Cu-Zn superoxide dismutase; SOD), and catalase (CAT) were evaluated. Results: The standardized grape suspension up-regulated both SOD and CAT activity of bladder muscle and mucosa in control animals. There were few differences in the grape suspension treated animals after ischemia, and in general the activities decreased following I/R. Conclusions: Increases of SOD and CAT activity in control animals as a result of grape suspension suggest a greater antioxidant capacity. This increase in the antioxidant defense system may explain the increased protection of grape suspension in the face of ischemia and I/R. However, the activities of both enzyme systems decreased in the smooth muscle subjected to I/R showing that reperfusion damages these systems probably via oxidation damage to the enzymes themselves.