Comparative biochemical responses and antioxidant activities of the rabbit urinary bladder to whole grapes versus resveratrol.

The objective of this study is to compare the antioxidant activity of a whole-grape suspension with the antioxidant activity or pure resveratrol on the effect of hydrogen peroxide (H2O2) on malondialdehyde (MDA) generation, choline acetyltransferase (ChAT) activity, calcium ATPase activity, and sarcoendoplasmic reticular ATPase (SERCA) of the male rabbit urinary bladder. MDA was used as a model for the effect of H2O2 on lipid peroxidation. ChAT, SERCA, and calcium ATPase were evaluated based on their importance in urinary bladder physiology and pathology. Four male rabbit bladders were used. Each bladder was separated into muscle and mucosa, frozen under liquid nitrogen and stored at -80 °C for biochemical evaluation. The effect of H2O2 on the enzymes listed above was determined in the presence and absence of either resveratrol or a whole-grape suspension. (1) Resveratrol was significantly more effective than the grape suspension at protecting the bladder muscle and mucosa against peroxidation as quantitated by MDA formation. (2) The grape suspension was significantly more effective at protecting ChAT activity against oxidative stress of the muscle than resveratrol. (3) Neither the grape suspension nor resveratrol were particularly effective at protecting the bladder muscle or mucosa calcium ATPase or SERCA against oxidative stress. (4) ChAT was significantly more sensitive to oxidative stress than either calcium ATPase or SERCA. These data support the idea that the grape suspension protects the mitochondria and nerve terminals to a significantly greater degree than resveratrol which suggests that the activities of the grape suspension are due to the combination of active components found in the grape suspension and not just resveratrol alone.

Effect of hydrogen peroxide on contractility and citrate synthase activity of the rabbit urinary bladder in the presence and absence of resveratrol and a whole-grape suspension.

One etiology related directly to obstructive urinary bladder dysfunction is ischemia/reperfusion resulting in significant oxidative stress to the bladder. Grapes, a natural source of antioxidants, have been proven effective in preventing obstructive and ischemic bladder dysfunction. Many investigators believe that resveratrol is the primary active antioxidant ingredient in grapes. We compared the ability of a whole-grape suspension with pure resveratrol in their ability to protect the bladder from in vitro oxidative stress mediated by hydrogen peroxide (H2O2). Four male rabbit bladders were used. Two strips from each bladder were incubated in the presence of 1 mg/mL grape suspension for 30 min, another two strips were incubated in the presence of 1 mg/mL resveratrol solution, and the last two strips were incubated in the presence of 1 mg/mL sucrose/and fructose as controls. The rest of the bladder was separated into muscle and mucosa, frozen and stored for biochemical evaluation. (1) Chemically, resveratrol has about 20 times the antioxidant capacity of the grape suspension. (2) The grape suspension had significant protective effects when the rate of tension was quantitated at all concentrations of H2O2, while the resveratrol had no effect. (3) Citrate synthase activities of the muscle and mucosa were significantly protected by the grape suspension but not by resveratrol. These data demonstrate that the grape suspension protects the mitochondria to a significantly greater degree than resveratrol, which suggests that the antioxidant activities are due to the combination of active components found in the grape suspension and not just resveratrol.

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.

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.

Kohki tea protects the rabbit bladder from ischemia/reperfusion-induced contractile dysfunction.

OBJECTIVES: Results of several studies indicated that ischemia/reperfusion is an etiological factor in obstructive bladder dysfunction. Kohki tea pretreatment was shown to reduce the dysfunctions induced by partial outlet obstruction in rabbits. The current study was designed to determine if pretreatment of rabbits with Kohki tea could prevent the contractile dysfunctions induced by bilateral ischemia followed by reperfusion. METHODS: New Zealand white rabbits were separated into several groups; one half of each group was pretreated by oral gavage for 3 weeks with Kohki tea and the other half was treated with vehicle (water). Experimental groups were subjected to bilateral ischemia for either 1 or 3 h followed by reperfusion for either 1 h or 1 week (4 groups). The results from the experimental groups were compared to the groups of rabbits receiving sham operations. RESULTS: Under all experimental conditions, Kohki tea significantly reduced the contractile dysfunctions induced by ischemia and ischemia followed by reperfusion. CONCLUSIONS: This data is consistent with the concept that Kohki tea acts by protecting the bladder smooth muscle and mucosa from cellular damage caused by ischemia/reperfusion.

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

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

Coenzyme Q10 protect against ischemia/reperfusion induced biochemical and functional changes in rabbit urinary bladder.

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.

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

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

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.

Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: response to a standardized grape suspension.

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.

Oral Kohki Tea and its protective effect against in vitro ischemic damage to the bladder.

AIMS: Results of several studies indicate that ischemia/reperfusion (I/R) is an etiological factor in the contractile dysfunctions induced by partial bladder outlet obstruction in animal models. In support of this hypothesis, pretreatment of rabbits with Kohki Tea (Engelhardtia chrysolepis), a Japanese herbal drink very high in antioxidant activity, significantly reduced the contractile dysfunctions induced by partial outlet obstruction. The current study was designed to determine if pretreating rabbits with Kohki Tea could protect the bladder against the contractile damage induced by in vitro ischemia followed by re-oxygenation. METHODS: Forty-eight New Zealand White rabbits were separated into two groups of 24; Group 1 was pretreated by oral gavage for 3 weeks with Kohki Tea and Group 2 received vehicle (water). Each rabbit was anesthetized with pentobarbital. The urinary bladder was rapidly removed and eight longitudinal muscle strips were cut from the bladder body. Each strip was mounted in a separate 15-ml bath containing Tyrode's solution with glucose (1 mg/ml) and maintained at 37 degrees C. All strips were equilibrated for 30 min with a gas mixture of 95% O2 and 5% CO2. At the end of this period of time, all strips were stimulated with field stimulation (FS) carbachol and KCl. After the last wash, the aeration was changed to hypoxic mixture (nitrogen-CO2) without glucose. At the end of 2 hr, the aeration was changed back to the normal 95% O2 and 5% CO2, and glucose was added to the buffer. After 1 hr of re-oxygenation, a second set of stimulations was performed. In order to represent hyperreflexia, the strips were stimulated at 32-Hz FS at 5-min intervals during the hypoxic period in half of the in vitro experiments. RESULTS: The results showed that Kohki Tea pretreatment protected the bladder's response to FS from the detrimental effects of repetitive stimulation and the detrimental effects of both in vitro ischemia and repetitive stimulation on the contractile responses to carbachol and KCl. CONCLUSIONS: These data are consistent with the concept that Kohki Tea acts by protecting the bladder from cellular damage caused by hypoxia and the generation of free radicals.

Effect of oral Kohki tea on bladder dysfunction induced by severe partial outlet obstruction.

PURPOSE: Extracts of the leaves of Engelhardtia chrysolepis, a subtropical plant that grows wild in southern China, have been used medicinally in east Asia for hundreds of years. A standard extract named Kohki tea (Maruzen Pharmaceuticals, Onomichi City, Japan) is sold over the counter in Japan as a sweet tea shown to confer many beneficial effects on general health and well-being. The tea contains strong antioxidants, including several dihydroflavonol glycosides. The results of previous studies show that natural products with antioxidant activities provide protective effects on the bladder of rabbits with partial outlet obstruction. We determined in vivo and in vitro whether oral pretreatment of rabbits with Kohki tea protects the bladder from dysfunction induced by partial outlet obstruction. MATERIALS AND METHODS: A total of 28 New Zealand White rabbits were separated into 4 groups of 7 each. Rabbits in groups 1 and 2 were treated by gavage with 100 mg./kg. Kohki tea daily in distilled water, while those in groups 3 and 4 were given distilled water. After 4 weeks of daily oral administration each rabbit was sedated, the bladder was catheterized and cystometry was performed at a filling rate of 1 ml. per minute. At the completion of cystometry the rabbits were immediately anesthetized. Moderate outlet obstruction was created in groups 1 and 3, and sham surgery was performed in groups 2 and 4. Treatment was continued for an additional 4 weeks, when each rabbit was sedated and cystometry was repeated. After cystometry the bladder was exposed through a midline incision, excised, weighed and 4 strips of bladder body were cut for contractility studies. The balance of the bladder was separated between smooth muscle and mucosa by blunt dissection, frozen in liquid nitrogen and stored at -70C for biochemical analyses. RESULTS: Partial outlet obstruction stimulated similar increases in the bladder weight of all obstructed rabbits. Partial outlet obstruction resulted in a significant decrease in bladder compliance in all obstructed animals. However, the bladder of obstructed rabbits given Kohki tea were significantly more compliant than those given water. Voiding pressures in the control group and the obstructed group given distilled water were approximately equal, while obstructed rabbits given Kohki tea showed significantly higher maximal voiding pressure. The contractile responses to all forms of stimulation were reduced by obstruction to a significantly greater degree in the rabbits not given tea than in those given tea. Sarcoplasmic reticulum Ca2+-adenosine triphosphatase enzyme activity of the bladder was significantly reduced in obstructed rabbits given vehicle but activity was not reduced in obstructed rabbits given Kohki tea. CONCLUSIONS: Kohki tea had a significant protective effect on bladder function, contractile responses and bladder biochemistry in rabbits with moderate to severe partial outlet obstruction.