Estrogen replacement is protective to the effect of in vitro hypoxia on female rabbit bladder and pelvic floor contractile response.

Purpose: To explore the effect of estrogen replacement on pelvic floor and bladder contractile response to electrical field stimulation, following in vitro hypoxia in an animal model of surgical menopause. Materials and Methods: Twelve female adult rabbits were divided into three groups: control, ovariectomy, and ovariectomy with estradiol replacement. At 4 weeks animals were euthanized. Bladder, coccygeus, and pubococcygeus were isolated. Tissues were equilibrated with oxygenated Tyrodes containing glucose and stimulated with electrical field stimulation. Tissues were then stimulated under hypoxic conditions for 1 hour using nitrogenated Tyrodes without glucose. Tissues were then re-oxygenated for 2 hours and stimulated. Results: Pelvic floor required 10 times the stimulation duration (power) to achieve maximum contraction at 2 g baseline tension (10 ms duration) when compared to bladder (1 ms duration). Maximal tension generated was significantly greater for bladder than pelvic floor. Coccygeus and pubococcygeus were significantly less sensitive to the effects of hypoxia and had stable contractile response to field stimulation throughout the hour of hypoxia. Hypoxia resulted in progressive and rapid decline of bladder contractile strength. Following hypoxia, pelvic floor contractile recovery was superior to bladder. Improvement in the contractile response of both bladder and pelvic floor, during the period of post-hypoxia re-oxygenation, was significantly greater in ovariectomy animals treated with estradiol replacement. Conclusions: Replacement of estradiol at time of ovariectomy reduced oxidative stress on tissue and was protective to the effects of hypoxia on pelvic floor and bladder contractile function.

The effect of α-and δ-tocopherol-lipoic acid ester co-drugs on the response of the rabbit bladder to in vitro ischemia/reperfusion.

OBJECTIVE: Obstructive bladder dysfunction (OBD) caused by benign prostatic hyperplasia is a common medical problem in ageing men. As the prostate enlarges and compresses the urethra, the bladder wall thickness and the bladder is termed "compensated" because its function is still relatively normal. Subsequently, bladder function begins to fail and this change is termed "decompensation." The extent of decompensation progresses from mild through severe. Bladder decompensation is mediated by cyclical ischemia followed by reperfusion (I/R) resulting in an increased generation of free radicals and oxidative stress. Previous studies demonstrated that both vitamin E (tocopherol) and alpha-lipoic acid (LA) showed significant antioxidant activity in experimental urinary bladder oxidative stress models. We hypothesized that co-drugs derived from these antioxidants would result in enhanced antioxidant activity relative to either individual compound for the treatment of oxidative stress in the lower urinary tract. MATERIAL AND METHODS: Two ester co-drugs of TOC and LA, tocopherol ester (α-TOCE) and δ-TOCE were synthesized. Six adult male New Zealand White (NZW) rabbits were divided into two groups of three rabbits each. Eight full thickness strips from each rabbit bladder were taken for in vitro I/R experiments. The strips from the first set were control rabbits (24 strips). Six strips were not incubated, while the remaining strips were incubated in α-TOCE dissolved in 1% (n=6) or 2.5% ethanol (n=6) solutions. These strips were not subjected to in vitro I/R. The strips from the second set were processed as follows: 6 strips were not incubated, while the remaining strips were incubated in α-TOCE dissolved in 1% (n=6) or in δ-TOCE dissolved in 2.5% ethanol. These strips were subjected to 1 hour in vitro ischemia followed by two hours reperfusion. RESULTS: Preliminary studies demonstrated that neither antioxidant had any effect on the contractile responses to 1% or 2.5% ethanol. Neither antioxidant had any effect on the control contractile responses. Both antioxidants protected the tissue from the initial effects of ischemia. Both antioxidants had significant protective effects on the contractile responses to all forms of stimulation after the reperfusion period. CONCLUSION: Incubation with both antioxidants had similar protective effects on responses both to ischemia and to reperfusion.

Effects of Ganoderma Lucidum shell-broken spore on oxidative stress of the rabbit urinary bladder using an in vivo model of ischemia/reperfusion.

Oxidative stress plays an important role in specific disease pathophysiology and the aging process. In the history of human kind, many herbs were utilized for disease prevention and anti-aging treatment. However, there are few direct evidences provided by modern laboratory technology. The current study was designed to evaluate Ganoderma Lucidum's (GL) ability to reduce the damage from in vivo ischemia/reperfusion (I/R) using a rabbit model of I/R that has been effectively utilized to prove the effects of drugs and supplements to reduce oxidative stress. Urinary bladder dysfunction secondary to benign prostatic hyperplasia (BPH) is a major affliction of aging men. One of the major etiologies of obstructive bladder dysfunction (OBD) is oxidative stress induced by I/R. Pharmaceutical studies and clinical research have proven that GL is useful in helping to prevent certain types of pathology and also helpful in prolonging human life in part by acting as an antioxidant. Using an in vivo model of I/R, we have investigated the ability of GL to protect bladder function from oxidative damage mediated by I/R. Our studies demonstrated that ischemia followed by reperfusion resulted in a significant decrease in bladder compliance and decreases in the contractile responses to a variety of forms of contractile stimulation. Pretreatment of rabbits with Ganoderma Lucidum prior to subjecting the rabbits to I/R completely inhibited the negative effects of I/R on both the compliance and contractile responses. These results demonstrate that Ganoderma provides excellent protection of bladder function following I/R (oxidative stress).

A Novel Cystometric Model of Pelvic Floor Dysfunction After Rabbit Pelvic Floor Noxious Electrical Stimulation.

OBJECTIVES: Although a relationship between pelvic floor dysfunction and lower urinary tract symptoms is described in the literature, the mechanism and pathways need further characterization. We developed an animal model of pelvic floor dysfunction after noxious stimulation of the pubococcygeus (PC) muscle. METHODS: Fifteen female adult rabbits were evaluated with cystometry (CMG) and electromyography (EMG) recordings from the PC muscle. Cystometry/EMG was performed before and after treatment animal (n = 11) received noxious pelvic floor electrical stimulation through the PC EMG electrode, and controls (n = 4) underwent sham needle placement. Two animals underwent S3 dorsal rhizotomy to demonstrate that the observed results required afferent innervation. RESULTS: Voiding changes were demonstrated in 9 of 11 rabbits after stimulation. Most of the rabbits (7/9) exhibited a prolonged-dysfunctional voiding pattern with larger capacity (mean, 17 mL [SEM, ±8 mL]), longer intercontractile interval (227% [SEM, ±76%]) and duration (163% [SEM, ±20%]), and increased postvoid residual (24 mL [SEM, ±6 mL]). The remaining dysfunctional rabbits (2/9) exhibited an overactive-dysfunctional voiding pattern with lower capacity (-26 mL [SEM, ±6 mL]), shortened intercontractile interval (16% [SEM, ±9%]) and duration (56% [SEM, ±30%]), and decreased postvoid residual (-27 mL [SEM, ±6 mL]). Nonresponder rabbits (2/11) were relatively unchanged in their micturition cycles after stimulation. Rhizotomy animals were acontractile and filled until overflow incontinence occurred. CONCLUSIONS: Using noxious electrical stimulation of the pelvic musculature, we were able to produce an animal model of pelvic floor dysfunction in most rabbits as hallmarked by a larger bladder capacity, an increased intercontractile interval, and prolonged contraction duration.

Noxious electrical stimulation of the pelvic floor and vagina induces transient voiding dysfunction in a rabbit survival model of pelvic floor dystonia.

PURPOSE: Existing data supports a relationship between pelvic floor dysfunction and lower urinary tract symptoms. We developed a survival model of pelvic floor dysfunction in the rabbit and evaluated cystometric (CMG), electromyographic (EMG) and ambulatory voiding behavior. MATERIALS AND METHODS: Twelve female adult virgin rabbits were housed in metabolic cages to record voiding and defecation. Anesthetized CMG/EMG was performed before and after treatment animals (n=9) received bilateral tetanizing needle stimulation to the pubococcygeous (PC) muscle and controls (n=3) sham needle placement. After 7 days all animals were subjected to tetanizing transvaginal stimulation and CMG/EMG. After 5 days a final CMG/EMG was performed. RESULTS: Of rabbits that underwent needle stimulation 7 of 9 (78%) demonstrated dysfunctional CMG micturition contractions versus 6 of 12 (50%) after transvaginal stimulation. Needle stimulation of the PC musculature resulted in significant changes in: basal CMG pressure, precontraction pressure change, contraction pressure, interval between contractions and postvoid residual; with time to 3rd contraction increased from 38 to 53 minutes (p=0.008 vs. prestimulation). Vaginal noxious stimulation resulted in significant changes in: basal CMG pressure and interval between contractions; with time to 3rd contraction increased from 37 to 46 minutes (p=0.008 vs. prestimulation). Changes in cage parameters were primarily seen after direct needle stimulation. CONCLUSIONS: In a majority of animals, tetanizing electrical stimulation of the rabbit pelvic floor resulted in voiding changes suggestive of pelvic floor dysfunction as characterized by a larger bladder capacity, longer interval between contractions and prolonged contraction duration.

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 partial bladder outlet obstruction and reversal on rabbit bladder physiology and biochemistry: duration of recovery period and severity of function.

OBJECTIVES: To use a rabbit model of partial bladder outlet obstruction (BOO) to investigate the point at which obstructive bladder dysfunction becomes irreversible. METHODS: Partial BOO was induced in New Zealand White rabbits. It was then reversed and the rabbits were allowed to recover for 4, 8 or 12 weeks. Both at the time of reversal and at the end of the study, the rabbits were grouped according to bladder decompensation level (mild, intermediate or severe) based on bladder mass (weight). RESULTS: A strong correlation was observed between the production and distribution of collagen and the reduction of smooth muscle contractile function. We found that only in the bladders that were severely decompensated at the time of reversal did collagen levels not decrease. CONCLUSION: The data show that recovery of function after reversal of partial BOO is directly related primarily to collagen levels at the time of reversal.

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.

Effect of estrogen and ovariectomy on response of the female rabbit urinary bladder to two forms of in vitro oxidative stress.

INTRODUCTION AND HYPOTHESIS: There are several lower urinary tract dysfunctions (LUTD) that are more common in women than in men including incontinence, interstitial cystitis, and recurrent urinary tract infection. There is increasing evidence that these dysfunctions are associated with reduced blood flow, ischemia, hypoxia, and reperfusion resulting in free radical generation and oxidative damage. The goal of the current study was to determine if the level of circulating estrogen affects the response of the bladder muscle and mucosa to two in vitro models of oxidative stress: Incubation in the presence of hydrogen peroxide (H(2)O(2)) is the first model; the second is ischemia followed by reperfusion which results in the direct production of damaging free radicals. The motivation for this study is the current literature linking female-related LUTD with oxidative stress. METHODS: Eighteen female New Zealand white rabbits were divided into three groups: control, ovariectomized, and ovariectomized receiving continuous estrogen. Eight bladder strips from each of three rabbits per group were taken for in vitro ischemia/reperfusion (I/R) physiological experiments, while eight strips from the three remaining rabbit bladders per group were taken for in vitro H(2)O(2) experiments. All tissue was analyzed for total antioxidant activity (AA) and malondialdehyde (MDA) levels. In addition, the organ bath buffer was also analyzed for AA. RESULTS: In vitro H(2)O(2) was found to target the nerve, muscarinic receptor, and membrane equally causing more damage to bladder tissue than in vitro I/R. Ovariectomy resulted in lower contractility and higher lipid peroxidation. However, estrogen supplementation following ovariectomy protected the bladder against both models of oxidative stress by maintaining contractile responses to stimulation and decreasing lipid peroxidation. CONCLUSIONS: The primary conclusion from this study is high estrogen protects the bladder from oxidative stress, whereas low estrogen makes the bladder more susceptible.

The effect of in vitro oxidative stress on the female rabbit bladder contractile response and antioxidant levels.

Introduction. There are several bladder dysfunctions that are associated with oxidative stress to the urinary bladder. Two experimental models are known to cause this type of bladder damage. The first is direct oxidative damage caused by hydrogen peroxide (H2O2). The second is oxidative damage caused by ischemia followed by reperfusion (I/R). The specific aim of this study is to directly compare these two models of oxidative stress. Methods. Six adult female NZW rabbits were divided into two groups of three rabbits each. Eight full thickness strips from three rabbit bladders were taken for in vitro ischemia/reperfusion physiological analysis, while eight strips from three rabbit bladders were taken for in vitro H2O2 physiological analysis. All tissue was analyzed for total antioxidant activity (AA) and malondialdehyde (MDA) levels. In addition, samples of the water baths were also analyzed for AA. Results. In vitro I/R reduced the response to field stimulation (FS) to a significantly greater extent than the inhibition of the response to carbachol. In vitro H2O2 decreased all responses to approximately the same degree. Total AA levels at higher concentrations of H2O2 for all bath fluids were significantly higher than controls. MDA levels were significantly elevated in both models of oxidative stress.

The effect of partial outlet obstruction on calpain and phospholipase-2 activities: analyzed by severity and duration.

In an attempt to better understand the two pathways that lead to bladder decompensation following partial obstruction in rabbits one of which is caused by calcium-activated enzymes and the other by oxidative stress, calpain and phospholipase A2 (PLA2) biochemical assays were conducted to see how bladder decompensation is mediated by these two calcium-activated enzymes. Partial outlet obstructions of varying durations (4, 8, and 12 weeks plus controls) were performed on 32 New Zealand white rabbits. The rabbits were also grouped by severity: control, mild, intermediate, and severe. The activities of Calpain and PLA2 on the muscle tissue of the bladders were analyzed. A stronger correlation was seen between activities and severities as opposed to between activities and durations for both PLA2 and calpain. The activity for PLA2 increased dramatically from control to mild and then stayed constant for both intermediate and severe obstructions. Calpain activity increased steadily from control to mild to intermediate to severe. Based on the increase in levels of the calcium-dependent enzymes, it was clearly shown that calcium levels increased in all stages of bladder decompensation most notably with the mild obstructions. Based on previous studies in which nitrotyrosine and dinitrophenol levels did not increase in mildly obstructed rabbits, the calcium overload pathway may predominate in mild decompensation because cells in mildly obstructed bladders are better able to cope with oxidative stress than increased calcium levels.

Oxidative effect of several intravenous iron complexes in the rat.

The objective of this study was to compare the oxidative stress induced in rat internal organs by the administration of the following clinically used intravenous (IV) iron (Fe) containing compounds: iron sucrose (IS), iron dextran (ID), ferric carboxymaltose and ferumoxytol. Groups of six adult rats received 1 mg/kg of each compound weekly for 5 doses. Seven days following the last dose, animals were euthanized and tissue samples of heart, lung, liver, and kidney were obtained, washed in warmed saline and frozen under liquid nitrogen and stored at -80 °C for analysis for nitrotyrosine (NT) and dinitro phenyl (DNP) as markers of oxidative stress. All tissues showed a similar pattern of oxidative stress. All Fe products stimulated an increase in the tissue concentration of both NT and DNP. In general, DNP was stimulated significantly less than NT except for IS. DNP was stimulated to an equal degree except for ID where NT was significantly higher than the NT concentrations in all other Fe compounds. ID produced over 10-fold the concentration of NT than any other Fe. IV Fe compounds present a risk of oxidative stress to a variety of internal organs. However, we found that IS was the least damaging and ID was the worst.

Effect of severity and duration of bladder outlet obstruction on catalase and superoxide dismutase activity.

OBJECTIVES: To study the effects of partial bladder outlet obstruction on the cell's anti-oxidant defense mechanisms, superoxide dismutase and catalase, in order to elucidate how the bladder responds to oxidative stress. METHODS: Four groups of eight rabbits were subjected to partial bladder outlet obstruction for 4, 8 and 12 weeks. Eight sham rabbits were used as the control group. The bladders were removed under anesthesia, and the muscle and mucosa were separated, frozen and stored at -80°C for analysis. Superoxide dismutase and catalase assays were carried out on these tissues. The groups were also categorized by severity (mild, intermediate and severe) of decompensation, as well as duration. RESULTS: When separated by duration, catalase activity of the mucosa was significantly higher in the control and the 12-weeks obstructed rabbits. This activity was lower than the control in the 4- and 8-weeks obstructed rabbits. When separated by severity, catalase activity of the mucosa was significantly higher and severely decompensated than the muscle in the controls. When separated by duration or severity, superoxide dismutase activity of the muscle was significantly lower than controls for all obstructed rabbits. The activities of both superoxide dismutase and catalase were significantly reduced in the severely decompensated bladder smooth muscle, but not in the 12-weeks obstructed bladder smooth muscle. CONCLUSIONS: Partial bladder outlet obstruction has significant effects on the activity of both superoxide dismutase and catalase in the bladder, with variations that are dependent on the severity and duration of the obstruction.

Partial outlet obstruction in rabbits: duration versus severity.

OBJECTIVES: Oxidative stress is a major etiology of obstructed bladder dysfunction. The major goal of the current study was to correlate the level of oxidative stress with both the severity and duration of obstruction. METHODS: A total of 32 New Zealand White rabbits were divided into four equal groups. Groups 1-3 received partial bladder outlet obstructions by standard methods and survived for 4, 8 or 12 weeks. Group 4 received sham surgery at the end of each time period, isolated strips were taken for contractility studies and the balance of the bladder was frozen as muscle and mucosa for quantification of nitrotyrosine and carbonyl-oxidized proteins derivatized into dinitrophenyl. For each duration, the eight rabbits were divided into three severity groups: mild, intermediate or severe decompensation. RESULTS: Contractile responses decreased in proportion to both severity and duration. The level of both oxidative products correlated to a much higher degree with the level of severity than the duration. There were significant decreases in the contractile responses in the mild decompensation group, whereas the level of derivatized into dinitrophenyl and nitrotyrosine of the muscle remained at control levels. This was not the case for the 4 weeks obstructed group. CONCLUSIONS: These findings suggest that the etiology for the mechanism of contractile dysfunction is not an oxidative stress.

Citrate synthase, sarcoplasmic reticular calcium ATPase, and choline acetyltransferase activities of specific pelvic floor muscles of the rabbit.

There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic-endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity.

Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis.

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

Calcium effects on superoxide dismutase and catalase of the rabbit urinary bladder muscle and mucosa.

PURPOSE: Superoxide dismutase (SOD) and catalase are two important antioxidant mechanisms that work together to reduce free radical damage. Intracellular free calcium in smooth muscle can change rapidly and many enzymes can be affected. The sensitivity of SOD and catalase activity to calcium was determined in both rabbit bladder smooth muscle and mucosa. MATERIALS AND METHODS: Calcium sensitivity was analyzed by determining SOD and catalase activity in muscle and mucosa at the following calcium concentrations: 0 (in the presence of 1 mM EGTA), 1 and 5 mM CaCl(2). RESULTS: SOD: EGTA resulted in increased SOD activity of bladder smooth muscle, whereas both 1 and 5 mM calcium significantly decreased SOD activity. EGTA had no effect on SOD activity of the mucosa whereas 1 and 5 mM calcium decreased SOD activity of the muscle. Catalase: 1 mM calcium resulted in decreased catalase activity of the muscle and no change in the activity of the mucosa, whereas 5 mM calcium resulted in increased catalase activity of the mucosa but no change in the activity of the muscle. DISCUSSION: Mucosa showed more SOD and catalase activity than the muscle. Both SOD and catalase showed differing sensitivities to EGTA and calcium.

The effect of antioxidants on the response of the rabbit urinary bladder to in vitro ischemia/reperfusion.

To evaluate the protective effects of two naturally occurring antioxidants, α-Lipoic acid and coenzyme Q10 on the response to in vitro ischemia of the rabbit urinary bladder. We measured free fatty acid (FFA) content, phospholipid (PL) content, malondialdehyde (MDA) levels, and phospholipase A(2) activity (PLA) of subcellular compartments. Twenty New Zealand White male rabbits were separated into four groups of five rabbits each. The in vitro whole bladders from Groups 1 and 2 received a 3 h incubation under normal oxygenated physiological conditions. The bladders were stimulated by field stimulation at 1 and 3 h. The bladders from groups 3 and 4 underwent 1 h incubation time under normal oxygenated physiological conditions. After 1 h, the bladders were stimulated with field stimulation. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations occurring at 5 min intervals during this time. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h reperfusion period after which the stimulation was repeated. The rabbits from groups 2 and 4 received α-Lipoic acid (10 mg/kg/day) + Coenzyme Q10 (3 mg/kg/day) by gavage for 4 weeks before the experiment. At the end of the experimental period, each bladder was opened longitudinally, and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and cytosol (supernatant) components. PL, FFA, MDA, and PLA were analyzed using standard biochemical techniques. Post-ischemic contractility only returned to 30% of control of the untreated group. However, post-ischemic contractility of the treated group returned to approximately 70% of control. PL loss in the muscle mitochondria and synaptosomes was prevented by antioxidant treatment, while the mucosal layer showed a significant drop in PL with antioxidants treatment. Administration of CoQ + LA significantly decreased MDA levels in both control and ischemic tissues in both the muscle and mucosal bladder layers, especially substantial in the microsomal and mitochondrial components. Treatment had variable effects on PLA(2) activity. Treatment of bladder dysfunction with antioxidants daily can be beneficial in man to prevent or delay the onset of progressive loss of bladder function especially that due to ischemic damage to mitochondrial and microsomal lipids. CoQ10 + LA can provide similar protection of the bladder muscle and mucosa against lipid oxidative stress as they have been shown to protect against protein oxidative damage.

The effect of in vitro ischemia/reperfusion on contraction, free fatty acid content, phospholipid content, and malondialdehyde levels of the rabbit urinary bladder.

To evaluate the effects of in vitro ischemia/reperfusion on contractile response to field stimulation (FS), free fatty acid (FFA) content, phospholipid (PL) content, and malondialdehyde (MDA) levels of the rabbit urinary bladder. There is significant evidence that ischemia/reperfusion injury is linked to obstructive bladder dysfunction secondary to men with benign prostatic hyperplasia (BPH). Twelve New Zealand White male rabbits were separated into two groups of six rabbits each. Each rabbit was euthanized, and the bladder was surgically removed intact for whole bladder incubation. The bladders in Group 1 received a 3-h incubation under normal oxygenated physiological conditions. These bladders received electrical field stimulation (32 Hz) after 1 and 3 h. The bladders associated with Group 2 received a 1-h incubation under normal oxygenated physiological conditions. At the end of this 1-h period, the bladders were subjected to FS. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium was changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations (32 Hz) occurring at 5-min intervals to represent overactive bladder dysfunction. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h period after which the stimulation was repeated. At the end of the experimental period, each bladder was opened longitudinally and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and supernatant (cytosol) components. PL, FFA, and MDA content were analyzed for each fraction using standard biochemical techniques. The bladder contractile responses decreased during the period of in vitro ischemia and returned to only 30% of control after reperfusion. In vitro ischemia/reperfusion showed the following: (1) There was a modest but significant decrease in the FFA content of the microsomes of the muscle and significant increases in the FFA content of the nuclei and mitochondria of the mucosa. (2) There were decreases in the PL content of the homogenate and microsomes of the muscle and decreases in the PL content of the homogenate, microsomes, and supernatant of the mucosa. (3) Significant increases were observed in the MDA levels of the homogenate, mitochondria, and microsomes of both the muscle and mucosa. The significant increases in the lipid peroxidation of the bladder smooth muscle are consistent with the marked decrease in the contractile ability of the bladder following ischemia/reperfusion. The specific increased lipid peroxidation of the mitochondrial and microsomal components is consistent with the specific dysfunctions of the mitochondria and innervations observed following I/R in earlier published studies. The marked increases in lipid peroxidation in the mucosa associated with the loss of PL and FFA from this component are consistent with the significant dysfunction in both the antiadherence and antipermeability properties of the mucosa and may play a major role in the symptomatic nature of I/R-linked diseases of the bladder.

Differential effects of coenzyme Q10 and α-lipoic acid on two models of in vitro oxidative damage to the rabbit urinary bladder.

PURPOSE: Partial bladder outlet obstruction (PBOO) in rabbits causes free radical production through ischemia and reperfusion within the bladder smooth muscle and mucosa. We had previously shown that pretreatment of rabbits with a combination of α-lipoic acid (αLA) and coenzyme Q10 (CoQ) protected the bladder from contractile and metabolic dysfunctions mediated by PBOO. In this study, we examined the ability of pretreatment with αLA and CoQ combination in rabbits to protect the bladder from contractile damage mediated by either hydrogen peroxide (H(2)O(2)) or in vitro ischemia-reperfusion (I/R) which represents two in vitro models of oxidative damage. METHODS AND MATERIALS: Eight adult male New Zealand white rabbits were pretreated with CoQ and αLA orally for four weeks. Eight adult male control rabbits were given vehicle. Eight full-thickness bladder strips were isolated from each of 4 treated and 4 control rabbit bladders, and a dose-response curve to H(2)O(2) (0.1-0.8%) was generated. Similarly, isolated strips of bladder from the remaining 4 control and 4 treated rabbits were subjected to 1 h of ischemia (no oxygen without glucose) followed by 2 h of incubation in oxygenated buffer with glucose. The effects on the contractile responses to field stimulation (FS) at 2, 8, and 32 Hz, carbachol, and potassium chloride (KCl) were determined. RESULTS: H(2)O(2) reduced the contractile responses to KCl and carbachol to a significantly greater degree than to FS, whereas I/R reduced the contractile responses to FS to a significantly greater degree than to KCl and carbachol. Pretreatment of the rabbits with the combination of CoQ and αLA significantly protected the bladder from the damaging effects of I/R, but had virtually no effect on the damaging effects of H(2)O(2). CONCLUSION: Although both H(2)O(2) and I/R are in vitro models of oxidative free radical damage to bladder smooth muscle, they have significantly different methods of action and different sensitivities to antioxidants.