Correlation of in vivo bladder blood flow measurements with tissue hypoxia.

AIMS: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: We have demonstrated that we can determine the severity of ischemia by surface measurement of blood flow.

Coenzyme Q10 diminishes ischemia-reperfusion induced apoptosis and nerve injury in rabbit urinary bladder.

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 1 and 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.

Estrogen administration attenuates bladder outlet obstruction induced oxidative stress in the female rabbit.

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.

The beneficial effect of coenzyme Q10 and lipoic acid on obstructive bladder dysfunction in the rabbit.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: The current study clearly demonstrates that coenzyme Q10 and alpha-lipoic acid supplementation can improve bladder function after outlet obstruction.

The immediate effect of nitric oxide on the rabbit bladder after ovariectomy.

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, premedicated 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.

Protein oxidation as a novel biomarker of bladder decompensation.

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.

The effect of ovariectomy and oestrogen therapy on the free fatty acid content, endogenous lipase activity, and phospholipid content of the rabbit urinary bladder.

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 beta-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.

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.

Novel alterations in superoxide dismutase and catalase activities in the female rabbit bladder subjected to hormonal manipulations.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Protective effect of Antrodia camphorata on bladder ischemia/reperfusion injury.

INTRODUCTION: 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). METHODS: 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. RESULTS: 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.

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

OBJECTIVES: 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). METHODS: 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. RESULTS: 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. CONCLUSION: This study showed that PTHrP is increased after partial bladder outlet obstruction and decreased after relieving the obstruction.

Effects of inosine on response to in vitro hypoxia in absence of substrate on bladder dysfunction in adult rats.

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.

The effect of ischemia/reperfusion on rabbit bladder--role of Rho-kinase and smooth muscle regulatory proteins.

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.

Effect of ischemia/reperfusion on bladder nerve and detrusor cell damage.

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.

The effect of 2- and 4-week ovariectomy on female rabbit urinary bladder function.

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) alpha and beta 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 alpha was significantly increased, whereas ROK beta 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.