Obstructed Rabbits Research Articles

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.

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

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. 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. 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. 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.

Treatment of Obstructive Bladder Dysfunction in Rabbits with Coenzyme Q10 and Alpha Lipoic Acid

Objectives: Partial bladder outlet obstruction (PBOO) is one of the major pathological effects of benign prostatic hyperplasia in men. It is well demonstrated that ischemia followed by reperfusion and the resulting generation of free radicals and oxidative damage to proteins occur in our rabbit model of PBOO and are involved in the observed progressive obstructive bladder dysfunction. The aim of the current project was to determine if treatment of obstructed rabbits with alpha lipoic acid and coenzyme Q10 can reverse the urodynamic, biochemical, and physiological responses to PBOO.Methods: Twenty adult NZW male rabbits were divided into four groups (n = 5 per group): control (sham surgical) animals with no treatment (group 1); control animals treated with coenzyme Q10 + alpha lipoic acid for 3 weeks beginning 4 weeks after surgery (group 2); 7‐week obstructed rabbits (group 3); and 7‐week obstructed rabbits treated for the last 3 weeks with the combination of the two drugs (group 4).Results: Bladder weight, compliance, volume at micturition and contractile response to all forms of stimulation were reduced following obstruction, but significantly improved after treatment. In addition, the level of reactive oxygen species and reactive nitrogen species damage was significantly reduced by treatment.Conclusion: Our results provide additional support for the beneficial effects of treatment of PBOO with strong antioxidants.

Effect of Age on the Role of Rho-Kinase in Short-term Partial Bladder Outlet Obstruction

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

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

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. 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. 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. 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.

The effect of tamsulosin on the response of the rabbit bladder to partial outlet obstruction

To determine if tamsulosin treatment prevents or decreases the incidence and severity of outlet obstruction-induced bladder dysfunction in rabbits. Male New Zealand White rabbits were treated with tamsulosin or vehicle for 4 weeks with treatments initiated 1 week prior to sham or obstruction surgery. Cystometry was done on anesthetized rabbits 21 days after surgery. The bladders were then removed, weighed, and prepared for in vitro whole bladder studies. Responses to 32 Hz field stimulation (FS), carbachol, phenylephrine, and KCl were measured. Obstruction resulted in a significant increase in bladder weight, which was unchanged by tamsulosin treatment and a significant increase in micturition pressure in the vehicle-treated group but not in the tamsulosin-treated group. Compliance was significantly decreased in both obstructed groups. The vehicle-treated obstructed rabbits had a very sharp increase in intravesical pressure as the bladder reached capacity; this was not seen in the tamsulosin-treated obstructed rabbits. Tamsulosin did not change the pattern of modifications in contractile responses induced by bladder outlet obstruction. In vitro responses of vehicle and tamsulosin-treated obstructed rabbit groups in this study were similar. A greater micturition pressure was found for the vehicle-treated obstructed group than for the tamsulosin-treated obstructed group, which was probably due to decreased urethral resistance in the latter. On a functional basis, the higher compliance at capacity and decreased micturition pressure in the tamsulosin-treated obstructed group would be considered beneficial for bladder function.

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction.

Regional differences in responses of rabbit detrusor to electrical and adrenergic stimulation: influence of outlet obstruction

To examine regional responses of control and obstructed rabbit detrusor strips to electrical and adrenergic stimulation, and determine whether outlet obstruction causes regional variations in blood flow throughout the detrusor, as the detrusor smooth muscle of the bladder body has previously been considered homogeneous in its pharmacological properties. Fourteen male rabbits had the bladder outlet surgically obstructed for 2 weeks and were compared with 10 unoperated control rabbits. Blood flow was measured with the bladder empty and at capacity, using fluorescent microspheres. Paired dorsal and ventral strips were harvested from the midline equatorial detrusor and electrically and adrenergically stimulated. Obstructed rabbits had significantly higher bladder capacities and bladder weights than control rabbits. Dorsal strips from both control and obstructed rabbits contracted in response to noradrenaline, whereas ventral strips relaxed. The addition of prazosin, a nonselective alpha1-adrenergic-receptor blocker, completely blocked the contraction in dorsal strips, but had no effect on responses of ventral strips. There was also a regional difference in response to electrical stimulation, with ventral strips generating significantly more tension than dorsal strips in both control and obstructed rabbits. There were no regional differences in detrusor blood flow. Obstruction resulted in significantly lower responses to all forms of stimulation, and significantly less blood flow throughout the detrusor. There are regional differences in adrenergic receptor function and response to electrical-field stimulation throughout control and obstructed rabbit detrusor, a region that was previously thought to be functionally homogeneous. These differences must be recognized and acknowledged to obtain accurate and reproducible data from in vitro studies of the bladder.

Enhancement of indomethacin-induced gastric damage by mouth breathing in rabbits

Previous studies have reported that mouth breathing is associated with respiratory acidosis. Regarding to the reports that renal elimination of weak acids such as indomethacin is pH dependent, this study was carried out to evaluate the role of mouth breathing on plasma level of indomethacin and indomethacin-induced gastric damage in rabbits. Mouth breathing was induced by surgical ligation of nostrils under general anesthesia. One day after the operation, arterial blood samples were collected for acid–base balance analysis and indomethacin was administered intraperitoneally in a single dose of 40 mg/kg. The animals were killed 4 h after indomethacin administration and blood samples were collected for spectrofluorometric determination of indomethacin in plasma. The results showed that indomethacin induces more severe gastric damage in nose obstructed rabbits compared with sham and unoperated (UNOP) animals. Acid–base analysis revealed a respiratory acidosis in nose obstructed rabbits and indomethacin level of plasma was significantly higher in nose obstructed animals in comparison with control rabbits. The study shows that mouth breathing can increase the potentiation of indomethacin-induced gastric mucosal damage that may be due to higher level of indomethacin in plasma of nose obstructed animals.

Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation.

We showed that partial obstruction of the rabbit bladder outlet caused decreases in detrusor blood flow that were directly proportional to the level of decompensation present. Bladder decompensation is characterized by decreases in detrusor contractility, mitochondrial function, and sarco/endoplasmic reticulum calcium ATPase (SERCA) activity in obstructed rabbits. The current study was designed to create bladder decompensation and to relate its characteristic dysfunctions to the presence or absence of hypoxia in the obstructed rabbit bladder wall. Tissue hypoxia was visualized immunohistochemically after administration of a hypoxia probe in vivo. Twelve New Zealand White rabbits were separated into two groups. The rabbits in group 1 received sham operations; the rabbits in group 2 received partial outlet obstructions by standard methods. Four weeks after surgery, each rabbit received an intraperitoneal injection of aqueous Hypoxyprobe-1, which forms protein adducts in cells having O(2) concentrations less than 14 microM. Two hours after injection, the rabbit was anesthetized and the bladder exposed through a midline incision. One full-thickness bladder strip was cut and immediately placed in fixative for immunohistochemical recognition and visualization of Hypoxyprobe-1-protein adducts. The remaining bladder was then excised, and three additional strips were cut for contractility studies. The remainder of the bladder was frozen for biochemical and slot-blot analyses. Bladder weight was increased fourfold after obstruction, and significant contractile and biochemical dysfunctions were observed that indicated an intermediate level of decompensation. Immunohistochemical visualization revealed focal areas of moderate to severe hypoxia in the detrusor smooth muscle (SM) and subserosal regions of these bladders. No hypoxia was observed in the obstructed bladder mucosa, consistent with the absence of biochemical dysfunction in this compartment, or in unobstructed bladders. Slot-blot analyses confirmed the presence of significant Hypoxyprobe-1-protein adducts in the detrusor of the obstructed bladder, whereas none were present in the control bladder detrusors. Partial outlet obstruction of rabbit bladders resulted in focal areas of moderate to severe hypoxia in the detrusor SM and subserosal regions concomitant with increased bladder mass, decreased contractile function, and selective metabolic dysfunctions of the SM consistent with an intermediate stage of decompensation. The metabolic characteristics of the normoxic mucosa were normal a were those of unobstructed bladders.

Effect of Oral Kohki Tea on Bladder Dysfunction Induced by Severe Partial Outlet Obstruction

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. 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. 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. Kohki tea had a significant protective effect on bladder function, contractile responses and bladder biochemistry in rabbits with moderate to severe partial outlet obstruction.

Effect of Oral Tadenan Treatment on Rabbit Bladder Structure and Function After Partial Outlet Obstruction

There is increasing evidence that the progressive dysfunction induced by partial outlet obstruction is mediated by ischemia-reperfusion, and bladder decompensation results from ischemia-reperfusion induced damage to the cellular and subcellular organelle membranes of nerve and smooth muscle, mitochondria and sarcoplasmic reticulum. Tadenan, an extract of Pygeum africanum, is a therapeutic prescribed in Europe to relieve symptoms of obstructive bladder dysfunction secondary to benign prostatic hyperplasia. There is excellent experimental evidence that Tadenan treatment of obstructed rabbits reduces and reverses the progression of bladder decompensation. We determined whether Tadenan therapy can reverse the morphological damage associated with obstructive dysfunction. A total of 36 male New Zealand White rabbits were separated into 6 groups of 6 each. Rabbits in groups 1 and 2 underwent sham operation. For 3 weeks beginning 2 weeks after sham operation group 1 was treated with vehicle and group 2 was treated with 30 mg./kg. Tadenan daily. Rabbits in groups 3 to 6 underwent partial outlet obstruction surgery. Two weeks after obstruction each rabbit was treated for 3 weeks with vehicle in group 3, and with 1, 10 and 30 mg./kg. Tadenan in groups 4, 5 and 6, respectively. After the completion of treatment cystometry was performed on each rabbit and isolated bladder strips were evaluated for contractile responses to field stimulation, adenosine triphosphate, carbachol and KCl. Separate strips were fixed for electron microscopy to determine the location and severity of cellular and subcellular membrane damage. Partial outlet obstruction resulted in reduced compliance, decreased responses of bladder strips to all forms of stimulation tested, and significant and extensive damage to cellular and subcellular organelle membranes consistent with an ischemia-reperfusion etiology. Daily 1 and 10 mg./kg. Tadenan treatments had little effect on the obstruction induced increase in bladder weight or the deleterious changes in bladder function and structure. However, treating obstructed rabbits with 30 mg./kg. Tadenan daily resulted in reduced bladder hypertrophy, improved compliance, improved contractile responses to nearly normal levels of isolated bladder strips to all stimuli tested and reversal of obstruction induced structural damage to cellular and subcellular organelle membranes. Tadenan treatment of obstructed rabbits resulted in a dose dependent improvement in bladder ultrastructure in parallel with improved bladder compliance and contractile responses of isolated strips to stimulation, providing support for the hypothesis that damage to cellular and subcellular organelle membranes mediates the contractile dysfunction induced by partial outlet obstruction.

Aspirin treatment improves bladder function after outlet obstruction in rabbits

Objectives. To examine whether bladder smooth muscle dysfunction after outlet obstruction could be altered by treatment with aspirin. Long-term outlet obstruction causes contractile and metabolic dysfunction of the bladder in vivo and in vitro. The evidence is growing that a decrease in bladder perfusion is an important cause of this phenomenon. The platelet aggregation inhibitor, acetylsalicylic acid (aspirin), has been used to improve perfusion of the heart for decades. Methods. Ten male New Zealand white rabbits were obstructed for 4 weeks. Five rabbits received no further treatment (Obs), and 5 rabbits received 2 mg/kg/day aspirin (Obs+aspirin), administered by an osmotic pump implanted subcutaneously 1 week before the surgical obstruction. The bleeding time was measured to confirm the effectiveness of the aspirin treatment. Three different control groups were created: sham-operated rabbits, unobstructed rabbits with pumps containing DMSO (vehicle), and unobstructed rabbits with pumps containing aspirin. The contractile responses of bladder strips to field stimulation, adenosine triphosphate, carbachol, and KCl were determined. A section of each detrusor tissue was fixed in formalin and used to determine the smooth muscle and collagen (connective tissue) volume fraction. Results. No differences were found in the bladder weights or responses to stimuli in the different control groups, which were therefore combined. Partial bladder outlet obstruction caused significant increases in the bladder weight of the obstructed animals (Obs+aspirin, 10.15 ± 0.87 g; Obs, 10.17 ± 0.88 g; and controls, 2.87 ± 0.21 g). The aspirin treatment increased the bleeding time from 1.7 ± 0.3 minutes to 3.3 ± 0.1 minutes. The responses to field stimulation were significantly reduced in all of the obstructed rabbits. However, the responses of the bladder strips from the Obs rabbits to field stimulation were impaired to a significantly greater degree than were those from the Obs+aspirin rabbits. The response to 32-Hz stimulation was reduced by 86% in the Obs group but by only 64% in the Obs+aspirin group. The responses to carbachol were significantly reduced by 62% in the strips from the Obs rabbits, but the responses of the strips from the Obs+aspirin rabbits were similar to the responses of the strips from the controls. The responses to KCl and adenosine triphosphate were reduced, although they just failed to achieve statistical significance using Bonferroni’s analysis. The ratio of smooth muscle and connective tissue shifted slightly toward smooth muscle after 4 weeks of obstruction, but no difference was found with or without aspirin treatment. Conclusions. Low-dose aspirin has a small but significant protective effect on the contractile dysfunction induced by bladder outlet obstruction in rabbits, although the increase in bladder mass was not altered. Bladders of the same weight showed improved responses to all forms of stimulation after pretreatment with aspirin. Already used by millions of patients with heart diseases, aspirin could be a useful protection against contractile dysfunction of the obstructed bladder.

Increased blood flow after catheterization and drainage in the chronically obstructed rabbit urinary bladder

Objectives. To determine the effect of drainage on rabbit bladder blood flow after 4 weeks of partial outlet obstruction. Previous studies have shown that catheterization and drainage of the urinary bladder in control rabbits resulted in a significant nitric oxide-induced increase of blood flow to the bladder. It was also shown that 4 weeks’ partial outlet obstruction caused a significant decrease in blood flow to the bladder. Methods. Male New Zealand White rabbits underwent partial outlet obstruction by standard methods. After 4 weeks, the blood flow to the bladder muscle and mucosa was determined by a microsphere technique. Within 1 to 2 minutes after transurethral catheterization and complete drainage of the bladder, the blood flow was again determined. Unobstructed animals served as controls. Four other control animals underwent a repetitive blood flow study during 10 minutes to determine the time frame of blood flow changes after drainage. Blood flow was also measured in 2 control rabbits after transurethral catheterization without drainage and in 2 control rabbits after drainage by suprapubic puncture. To exclude the possibility that increased intravesical pressure alters the blood flow measurements, the relationship between the intravesical volume and the bladder pressure was examined in the obstructed rabbits. Results. After drainage of the bladder, the blood flow to the bladder muscle increased 4.5-fold in the decompensated obstructed group (bladder weights greater than 15 g) and 2.5-fold in the compensated animals (bladder weights less than 5 g) and control animals. Blood flow to the mucosa followed the same pattern but without reaching significance. Blood flow returned to near baseline values within 5 minutes. Catheterization without drainage did not alter the blood flow. In contrast, drainage by puncture increased the blood flow significantly. Higher intravesical volumes increased the intravesical pressure slightly, but after opening the abdominal fascia, the intravesical pressure did not change with increasing volumes. Conclusions. Although the previously shown decreased blood flow to the bladder smooth muscle may be an etiologic factor in bladder contractile dysfunction secondary to partial outlet obstruction, the bladder does have the ability to increase the blood flow after drainage. This ability could be a compensatory and possibly protective mechanism after outlet obstruction.

IMPROVED CONTRACTILITY OF OBSTRUCTED BLADDERS AFTER TADENAN TREATMENT IS ASSOCIATED WITH REVERSAL OF ALTERED MYOSIN ISOFORM EXPRESSION

Tadenan is a plant extract from Pygeum africanum used in the treatment of benign prostatic hyperplasia, to protect the bladder from contractile dysfunction induced by partial bladder outlet obstruction (BOO). The aim of the present study was to determine whether the Tadenan-induced return of detrusor contractility affects the expression of myosin isoforms, which differ at the C-terminal (SM1 and SM2) and the N-terminal regions (SM-A and SM-B). Four groups of New Zealand White rabbits (3 to 5 kg., 4 to 6 rabbits per group) were either partially obstructed by ligation of the urethra (groups 1 and 2) or not obstructed (groups 3 and 4). After 2 weeks, rabbits from groups 2 and 4 received Tadenan in peanut oil (vehicle) orally at 100 mg. /kg./day for 3 weeks and rabbits in groups 1 and 3 received vehicle only. Rabbits were sacrificed and bladders were removed and weighed. Contractility studies were performed on isolated strips of detrusor and the remaining muscular layer from the bladder body was used to study the expression of myosin heavy chain (MHC) isoforms at mRNA (SM1, SM2, SM-A, and SM-B) and the protein (SM1 and SM2) levels by RT-PCR and SDS-PAGE analyses, respectively. Tadenan significantly reduced the effect of BOO on bladder mass. The diminished contractile response to field stimulation and carbachol secondary to urethral obstruction was significantly reversed by Tadenan treatment. The relative ratios for MHC isoforms were altered at the mRNA (SM2:SM1 and SM-A:SM-B) and protein (SM2:SM1) levels in obstruction. Upon treatment with Tadenan, the ratio of these isoforms returned to normal, as shown at the mRNA levels. In addition, the altered relative ratio of SM2:SM1 at the protein level also returned to nearly normal values after treatment. Improvement of obstruction-induced contractile dysfunction of the detrusor following treatment with Tadenan is associated with changes in the expression of myosin isoforms. The alteration in the expression of myosin isoforms associated with obstruction-induced hypertrophy is reversed close to normal in the detrusor smooth muscle from Tadenan-treated obstructed rabbits.

INTERMITTENT CATHETERIZATION LIMITS RABBIT BLADDER DYSFUNCTION IN RESPONSE TO PARTIAL OUTLET OBSTRUCTION

The initial response of the urinary bladder to outflow partial obstruction consists of distension, followed by a rapid increase in mass and concomitant functional adaptations. Subsequently, an indeterminate period of stabilized function and little increase in mass occurs (compensation). Finally, bladder mass again progressively increases, accompanied by deleterious changes in bladder morphology, biochemistry and pharmacology, and progressive loss of function (decompensation). The reported study was designed to determine whether limiting the level of bladder distension, using intermittent catheterization (IntCath), could protect the bladder from decompensation. Sixteen male New Zealand White rabbits were separated into 4 groups of 4 rabbits each: control, control with IntCath, obstructed, and obstructed with IntCath. IntCath was performed with the animals under inhalation anesthesia. An 8 Fr. catheter was inserted through the urethra into the bladder every 8 hours and the urine drained. After 14 days of obstruction, bladders were removed from all rabbits; longitudinal strips were cut from the bladder body and suspended in individual organ baths. Contractile responses to field stimulation (FS), KCl, ATP, and carbachol were measured. There were no significant differences between control rabbits and controls with IntCath in bladder weights, compliance, and contractile responses to all stimuli. Bladder weights of both obstructed groups increased significantly compared to those of both control groups. Bladder weights of obstructed rabbits increased to a significantly greater extent than did those of obstructed rabbits with IntCath. Bladder compliance in the obstructed group was significantly lower than compliance in both control groups and in obstructed rabbits with IntCath. Both obstructed groups exhibited significantly decreased contractile responses to FS, ATP and KCl, compared to control groups. The response of the obstructed group with IntCath to FS and carbachol was significantly greater than the responses of the obstructed group without IntCath. These findings show that limiting distension with IntCath reduces the magnitudes of the increased bladder mass, the loss of bladder wall elasticity (compliance), and the impaired contractile responses which occur secondary to outflow obstruction.

Beneficial effects of Tadenan therapy after two weeks of partial obstruction in the rabbit.

Tadenan (Debat Laboratories, France) is a plant extract used in Europe for the treatment of micturition disorders associated with benign prostatic hypertrophy (BPH). Prior studies demonstrated that pretreatment of rabbits with Tadenan significantly reduced the contractile dysfunction observed after 2 weeks of partial outlet obstruction. The specific aim of the present study was to determine the effect of Tadenan therapy following the creation of partial outlet obstruction. Two sets of experiments were performed: one with mild and the other with severe outlet obstruction. For both sets of experiments, male New Zealand rabbits (3-5 kg) were separated into 3 groups of 5 rabbits each. Each rabbit in groups 1 and 2 was obstructed using standard methodology. Rabbits in group 3 served as controls and did not receive any surgery. After 2 weeks, each rabbit in group 1 received Tadenan orally at 100 mg/kg/day for 3 weeks; each rabbit in group 2 received vehicle (peanut oil). After 3 weeks of treatment (5 weeks after partial outlet obstruction), rabbits were anesthetized and cystometries were performed. Immediately after cystometry, the rabbits were euthanized, the bladder rapidly removed, and 4 longitudinal strips prepared and mounted in individual baths for contractile studies. The contractile responses to field stimulation, carbachol, adenosine-5'-triphosphate (ATP), and potassium chloride (KCl) were determined, as follows: (1) Bladder mass approximately doubled in the mildly obstructed groups. Bladder mass increased significantly (3-5-fold) in the severely obstructed groups. (2) Cystometrograms from the mildly obstructed rabbits treated with peanut oil showed low compliance, whereas those of the mildly obstructed rabbits treated with Tadenan showed normal compliance. The cystometrograms of all severely obstructed rabbits showed low compliance. (3) Mild obstruction caused small but significant decreases in the contractile response to field stimulation that were reversed by Tadenan treatment. No changes were noted in response to bethanechol, ATP, and KCl stimulation. (4) Severe obstruction caused significant decreases in the response of bladder strips to field stimulation and bethanechol. Following Tadenan therapy, there was a significant improvement in the response to high-frequency field stimulation and a substantial improvement in the response to bethanechol (response equal to control). No changes were noted in response to ATP and KCl stimulation. In conclusion, Tadenan treatment reversed the bladder dysfunctions induced by mild partial outlet obstruction, and resulted in improved bladder function in the severe model of outlet obstruction. These studies are consistent with previous studies showing that Tadenan pretreatment protects the bladder against the development of contractile dysfunctions.

Effect of partial outlet obstruction on 14C‐adenine incorporation in the rabbit urinary bladder

Bladder outlet obstruction induces severe changes in urinary bladder function and metabolism. These changes are characterized by significant reductions in the ability of the in vitro whole bladder to generate pressure and to empty. Metabolically, partial outlet obstruction induces a shift from oxidative to anaerobic metabolism. The decreased oxidative metabolism is mediated in part by significant decreases in mitochondrial substrate metabolism, which in turn is correlated with decreased activity of 2 important mitochondrial enzymes: citrate synthase and malate dehydrogenase. The present study was designed to evaluate mitochondrial function by studying the incorporation of 14C-adenine into high-energy phosphates (ATP, AMP, and ADP). Mild partial outlet obstructions were created by surgically placing silk ligatures loosely around the bladder neck. The results of these studies demonstrate that after 60 min incubation in oxygenated medium containing glucose + uCi14C-adenine, 1) There was no significant differences in the total AMP, ADP, and ATP concentrations measured in bladders taken from controls, 7- and 14-day obstructed rabbits; 2) there was no effect of obstruction on either the concentration of 14C-AMP in the tissue or in the ratio of hot to cold AMP; and 3) there was a 50% decrease in the concentration of 14C-ADP and a 70% decrease in the concentration of 14C-ATP in the bladder smooth muscle obtained from obstructed tissue (from both 7- and 14-day obstructions) compared to concentration in the control bladder smooth muscle. These results confirm the previous finding that obstruction did not reduce the rate of incorporation of adenine to AMP within the obstructed bladder smooth muscle and extends these studies to identify a significant reduction in the synthesis of both ADP and ATP. These results support the hypothesis that partial outlet obstruction induce a major dysfunction in mitochondrial function, both in the ability to oxidize substrates and in the ability to generate ATP. Neurourol. Urodynam. 16:201–208, 1997. © 1997 Wiley-Liss, Inc.

Transcription of mitochondrial and mitochondria-related nuclear genes in rabbit bladder following partial outlet obstruction.

Using the rabbit model, we showed that partial outlet obstruction of the urinary bladder causes significant changes in the status and expression of the mitochondrial (mt) genetic system in bladder smooth muscle immediately after obstruction is initiated. Here we investigate quantitatively the severity of the mt genetic response to partial outlet obstruction in both short- and long-term obstructed rabbits. Based on previous functional studies, bladders with mass < 6 fold greater than control were considered compensated; bladders with mass > 6 fold that of control were considered decompensated. Analyses of DNA from compensated rabbit bladders showed that relative mt genome copy number decreased to 30% of control values. Transcript analyses for these samples showed that mt RNA levels increased 3 fold to compensate for lower template copy number. Analysis of decompensated bladders demonstrated that mt genome copy number increased to approximately 90% of control levels; mt transcripts progressively decreased in these samples by as much as 30 fold. In contrast, transcription of a mt-related nuclear gene decreased 3-9 fold in compensated bladders but increased 10-30 fold in decompensated bladders. Activity for the cytochrome oxidase complex, and for the mt enzyme citrate synthase, decreased steadily with increasing bladder hypertrophy. These data suggest that bladder dysfunction following partial outlet obstruction is mediated partly by a significant loss in mt and mt-related nuclear gene coordination.

Effect of partial outlet obstruction on 14C-adenine incorporation in the rabbit urinary bladder.

Bladder outlet obstruction induces severe changes in urinary bladder function and metabolism. These changes are characterized by significant reductions in the ability of the in vitro whole bladder to generate pressure and to empty. Metabolically, partial outlet obstruction induces a shift from oxidative to anaerobic metabolism. The decreased oxidative metabolism is mediated in part by significant decreases in mitochondrial substrate metabolism, which in turn is correlated with decreased activity of 2 important mitochondrial enzymes: citrate synthase and malate dehydrogenase. The present study was designed to evaluate mitochondrial function by studying the incorporation of 14C-adenine into high-energy phosphates (ATP, AMP, and ADP). Mild partial outlet obstructions were created by surgically placing silk ligatures loosely around the bladder neck. The results of these studies demonstrate that after 60 min incubation in oxygenated medium containing glucose + 1uCi14C-adenine, 1) There was no significant differences in the total AMP, ADP, and ATP concentrations measured in bladders taken from controls, 7- and 14-day obstructed rabbits; 2) there was no effect of obstruction on either the concentration of 14C-AMP in the tissue or in the ratio of hot to cold AMP; and 3) there was a 50% decrease in the concentration of 14C-ADP and a 70% decrease in the concentration of 14C-ATP in the bladder smooth muscle obtained from obstructed tissue (from both 7- and 14-day obstructions) compared to concentration in the control bladder smooth muscle. These results confirm the previous finding that obstruction did not reduce the rate of incorporation of adenine to AMP within the obstructed bladder smooth muscle and extends these studies to identify a significant reduction in the synthesis of both ADP and ATP. These results support the hypothesis that partial outlet obstruction induce a major dysfunction in mitochondrial function, both in the ability to oxidize substrates and in the ability to generate ATP.