Patrick Horan

Effect of Repetitive Stimulation on the Contractile Response of Rabbit Urinary Bladder Subjected to in vitro Hypoxia or in vitro Ischemia Followed by Reoxygenation

Purpose: We studied the effects of hypoxia followed by reoxygenation and an in vitro model of ischemia (hypoxia + substrate [glucose] deprivation) followed by reperfusion (reoxygenation + substrate replacement) on the contractile response of rabbit urinary bladder strips to nonrepetitive and repetitive field stimulation (FS), and correlated the results with the rate of lipid peroxidation. We view repetitive FS as a model for hyperreflexia. Methods: The effects of repetitive and nonrepetitive FS on the contractile responses of isolated strips of rabbit bladder to FS, carbachol, and KCl were determined in the presence of 3 different incubation media: O2 + glucose (normal physiological medium); N2 + glucose (in vitro hypoxia), and N2 – glucose (in vitro ischemia). Then, all strips were incubated for 1 h in normal physiological medium (‘reperfusion’) followed by a final stimulation; the resultant contractile responses were correlated with the level of lipid peroxidation as determined by malonedialdehyde (MDA) concentration. Results: Repetitive stimulation, a model of hyperreflexia, significantly increased the rate of development of contractile dysfunction in bladder tissue strips incubated in all 3 media as compared to nonrepetitive stimulation, which caused no degradation of the contractile response in normal physiological medium. The rate of development of contractile dysfunction was significantly greater in bladder tissue strips incubated in the in vitro ischemia medium (N2 – glucose) than in strips incubated in the hypoxia medium (N2 + glucose); which, in turn, was significantly greater than in those incubated in the normal physiological medium (O2 + glucose). Repetitive stimulation (‘hyperreflexia’) during all 3 incubation conditions resulted in increased [MDA] after reoxygenation or ‘reperfusion’. Incubation in in vitro ischemia buffer (N2 – glucose) followed by 1 h reoxygenation + substrate replacement stimulated lipid peroxidation to a significantly greater extent than did incubation in hypoxia buffer (N2 + glucose) followed by 1 h of reoxygenation; the level of lipid peroxidation, [MDA], paralleled the magnitude of the contractile dysfunctions present. Independent of the incubation medium, the magnitude of FS-induced contractile dysfunction after reoxygenation or ‘reperfusion’ was significantly greater than the magnitude of dysfunction in response to carbachol or KCl. Conclusions: The results demonstrate that the rate of contractile failure induced by in vitro ischemia is greater than that induced by in vitro hypoxia, and that the contractile response to FS is significantly more sensitive to both hypoxia and in vitro ischemia than is the contractile response to either carbachol or KCl. Repetitive stimulation (‘hyperreflexia’) increases the rate of contractile failure under all conditions tested, and the magnitude of the contractile failure may be due, in part, to the generation of free radicals and subsequent stimulation of lipid peroxidation upon reoxygenation or ‘reperfusion’.

Vascular response of the rabbit bladder to short term partial outlet obstruction

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall.In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged.Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process.All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.

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.

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.

Correlation of EGTA and Calcium- Blocking Agents on the Response of the Bladder to in vitro Ischemia

The effects of repetitive field stimulation (model of hyperrelexia) on the responses of isolated strips of rabbit urinary bladder to FS and carbachol were evaluated under a variety of incubation conditions. Compared to control conditions, 2 h of repetitive FS in normal, oxygenated Tyrode’s solution followed by incubation for 1 h with no stimulation resulted in a 50% decrease in contractile response to FS and a 30% decrease in the response to carbachol. Incubation in the absence of O2 and glucose was used as an in vitro model for ischemia. Repetitive stimulation during in vitro ischemia resulted in a significantly greater decrease in the contractile responses to FS and carbachol than did in vitro ischemia without repetitive stimulation. The magnitude of contractile dysfunctions in response to both stimuli were significantly reduced in the presence of EGTA (calcium chelator), diltiazem (calcium channel blocker) or pincidil (potassium channel opener). Incubation with thapsigargin (SR calcium uptake inhibitor) + ryanodine (SR calcium storage inhibitor) had no effect. The results of these studies indicate that inhibition of Ca2+ entry reduces the contractile dysfunctions induced by repetitive stimulation in the presence of in vitro ischemia. Inhibition of Ca2+i storage and release had no significant effect on the magnitude of contractile dysfunctions induced by repetitive stimulation an in vitro ischemia.

THE RESPONSE OF FETAL SHEEP BLADDER TISSUE TO PARTIAL OUTLET OBSTRUCTION

PURPOSE We characterized the response of fetal ovine bladder strips to stimulated contraction and relaxation, and compared this response to that of strips from the pregnant mother and those obtained after a short duration of fetal bladder outlet obstruction. MATERIALS AND METHODS Sham surgery or bladder obstruction was performed in fetal sheep at 90 days of gestation (term 147 days). Bladder tissue was obtained 3 and 5 days later. Isolated strips of full-thickness bladders from fetuses and pregnant females were mounted individually in Tyrodes solution containing glucose. The strips were subjected to electrical field stimulation. Alternate strips were stimulated by adding carbachol, adenosine triphosphate and KCl. Each strip stimulated by carbachol also underwent field stimulation in the presence of carbachol. Relaxation was also tested using isoproterenol and nitroprusside. RESULTS The response of isolated strips to field stimulation showed phasic contraction or biphasic response, consisting of initial phasic contraction followed by phasic relaxation and a return to control tension after the end of stimulation. In fetal bladder strips field stimulation at all frequencies after carbachol stimulation produced phasic relaxation or a biphasic response with an initial relaxation phase followed by phasic contraction. This field stimulated relaxant response was not present in adult female bladder strips. In addition, field stimulation stimulated relaxation was completely eliminated by pretreatment with N-nitro-L-arginine-methyl ester, indicating that relaxation was nitric oxide mediated. The fetal responses to all forms of stimulation and relaxation were significantly greater than those of pregnant females. After 5 days or greater of obstruction the responses to field stimulation were reduced significantly. In contrast, there were no significant differences in contractile responses to adenosine triphosphate, carbachol or KCl, or the relaxant response to field stimulation after obstruction. However, there was a significant reduction in relaxant responses to isoproterenol and nitroprusside. CONCLUSIONS In mid gestation sheep fetus contractile responses to field stimulation, adenosine triphosphate, carbachol and isoproterenol are well developed. The fetal ovine bladder shows a strong neuronal nitric oxide response that is not present in the pregnant mother and is maintained after short-term obstruction.

Sucrose diuresis protects rat bladder from outlet partial obstruction-induced contractile dysfunction.

OBJECTIVES Evidence is accumulating that bladder dysfunction caused by experimental partial obstruction of the bladder outlet can be reduced or reversed by treatment that results in upregulation of bladder function, even in the presence of obstruction. Inducing diuresis in rats or rabbits results in a significant increase in bladder mass and increased contractility in response to stimulation. The objective of the present study was to determine whether diuresis-induced amplification of bladder function in the rat could protect the bladder from contractile dysfunctions caused by partial outlet obstruction. METHODS Thirty-two rats were separated into four groups of 8 rats each. Groups 2 and 4 were fed 5% sucrose instead of water; groups 1 and 3 were fed only water. Three weeks later, partial outlet obstructions were created in groups 3 and 4. After 4 weeks of obstruction, all bladders were rapidly excised and cut into longitudinal strips; each strip was mounted in an isolated muscle bath for contractile studies. RESULTS Sucrose-induced diuresis caused a moderate but significant increase in bladder mass. Partial outlet obstruction stimulated significant increases in bladder mass in both water-drinking and sucrose-drinking groups; the bladder mass of sucrose-drinking rats, however, increased less than that of water-drinking rats. In water-drinking rats, partial outlet obstruction resulted in significantly decreased bladder strip contractility in vitro in response to field stimulation (1 to 32 Hz), carbachol (0.1 to 22 microM), and KCl (120 mM). After 3 weeks of sucrose-induced diuresis, partial obstruction of the rat bladder outlet did not result in decreased in vitro contractile responses to any form of stimulation applied. CONCLUSIONS Sucrose-induced diuresis caused an increase in bladder mass and an increase in contractile strength, consequently protecting the rat bladder from the contractile dysfunctions that usually follow partial outlet obstruction.

EFFECTS OF HYPOXIA, CALCIUM, CARBACHOL, ATROPINE AND TETRODOTOXIN ON THE FILLING OF THE IN-VITRO RABBIT WHOLE BLADDER

PURPOSE The urinary bladder stores urine at low intravesical pressure and empties the urine efficiently and completely. Bladder compliance is the property that allows the bladder to fill to near capacity without a large increase in intravesical pressure. The current study utilized an in vitro whole bladder model to determine the effects of hypoxia, alterations in extracellular calcium concentration, carbachol and atropine on bladder capacity and compliance. METHODS Mature male New Zealand White rabbits were used in this study. The urinary bladder was excised from the rabbit together with a short segment of proximal urethra and mounted in a 400 ml. isolated bath containing Tyrodes buffer. Bladder filling was started by opening the bladder to a saline reservoir placed 80 cm. above the bladder. Intravesical pressure, rate of pressure increase, rate of volume increase, and maximal volume were digitally recorded. The bladder filling was repeated while the whole bladder was subjected to hypoxia, high calcium concentration, the presence of EGTA, carbachol, atropine and tetrodotoxin respectively. RESULTS Results are summarized as follows: 1) Bladder filling was biphasic. There was an initial rapid rise in intravesical pressure followed by a slower rise. The final bladder volume averaged 46 ml. 2) Hypoxia significantly decreased the initial rate of the rise in intravesical pressure, increased the rate of bladder filling, and increased bladder volume by 43%. 3) Incubation of the bladder in the presence of EGTA also significantly decreased the initial rate of intravesical pressure rise, increased the rate of filling and increased bladder volume by 39%. 4) High concentrations of calcium increased the initial rate of rise in intravesical pressure. 5) Carbachol significantly increased the rate of intravesical pressure rise, decreased the rate of bladder filling, and decreased bladder volume. 6) Atropine and tetrodotoxin (TTX) had no effects on bladder filling. CONCLUSION In summary, alterations in muscle tone had significant effects on bladder capacity and compliance.

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

INTRODUCTION 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Effects of atropine, isoproterenol and propranolol on the rabbit bladder contraction induced by intra-arterial administration of acetylcholine and ATP

INTRODUCTION In the rabbit, both cholinergic and purinergic nerves mediate bladder contraction. Acetylcholine is the neurohumoral transmitter for the cholinergic nerves, and ATP is the neurohumoral transmitter for purinergic innervation. Beta-adrenergic stimulation mediates relaxation of the bladder. In the current study, we investigated the effects of atropine, isoproterenol and propranolol on the bladder contraction induced by intra-arterial administration of acetylcholine and ATP. METHODS Mature male New Zealand White rabbits were used in this study. A polyethylene catheter with an outer diameter of 0.043 inches was inserted through the rabbits right femoral artery until it reached the lower abdominal aorta. An 8 F catheter was inserted through the urethral orifice into the bladder and secured by tying a 2-0 silk ligature around the bladder neck. The catheter was connected to an infusion pump and a pressure transducer by a 3-way valve. After 15 ml. of saline was infused into the bladder, an intra-arterial administration of acetylcholine and ATP was infused and the change of intravesical pressure was quantitated and recorded with a Grass model 7D polygraph. The procedure was repeated after a 5-minute pretreatment with atropine, isoproterenol or propranolol. RESULTS The results are summarized as follows: 1) Baseline intravesical pressure was not altered by pretreatment with atropine. Pretreatment with atropine shifted the dose-response curve of acetylcholine to the right and the maximal response was reduced by 9%, 49% and 77% respectively with pretreatment with atropine 10(-8), 10(-7) and 10(-6) mole/kg. The dose-response curve of ATP was not significantly affected by pretreatment with atropine. 2) Baseline intravesical pressure was lowered by pretreatment with isoproterenol. Pretreatment with isoproterenol shifted both dose-response curves of acetylcholine and ATP rightward. The maximal response of acetylcholine was reduced by 10%, 26% and 37% respectively, and the maximal response of ATP was reduced by 6%, 31% and 43% respectively by pretreatment with isoproterenol 10(-8), 10(-7) and 10(-6) mole/kg. 3) Baseline intravesical pressure was not changed by pretreatment with propranolol. Both dose-response curves of acetylcholine and ATP were not significantly affected by pretreatment with propranolol. SUMMARY In conclusion, pretreatment with atropine inhibited acetylcholine-induced bladder contraction, but had no effect on ATP-induced contraction. Pretreatment with isoproterenol significantly inhibited both contractile stimulation by acetylcholine and ATP. Pretreatment with beta-adrenergic antagonist had no effect on the bladder contraction induced either by acetylcholine or by ATP. Thus, although beta-adrenergic stimulation is capable of significantly inhibiting the contractile responses to both cholinergic and purinergic stimulation, under normal conditions, sympathetic nerves do not modulate either cholinergic or purinergic stimulation.