Effect of diltiazem and pinacidil on the response of the rabbit urinary bladder to repetitive stimulation and in vitro ischemia

The effect of repetitive stimulation, in the presence and absence of diltiazem or pinacidil, on the contractile responses of isolated strips of rabbit bladder detrusor to field stimulation and carbachol, after 2 hr of incubation in a medium that serves as an in vitro model of ischemia (oxygen and substrate depleted Tyrode's solution), was determined. Our results are summarized as follows: a) The magnitude of the contractile dysfunctions after in vitro ischemia was enhanced by repetitive stimulation. b) Pre-incubation of isolated strips of detrusor with diltiazem (50 microM) inhibited the contractile responses to field stimulation (FS) and carbachol by 43 and 50%, respectively. Pinacidil (100 microM) inhibited the contractile responses to FS and carbachol by 37 and 32%, respectively. c) Neither diltiazem nor pinacidil protected the bladder strips against the effects of 2 hr of incubation in in vitro ischemia medium. However, d) both pinacidil and diltiazem reduced the level of contractile dysfunctions induced by repetitive stimulation. In conclusion, the contractile response to FS was significantly more sensitive to in vitro ischemia and repetitive stimulation than was the contractile response to carbachol. Both diltiazem and pinacidil protected the contractile responses to FS and carbachol from the degenerative effects of repetitive stimulation, but not from the effects of in vitro ischemia.     

Cyclical estrogen and free radical damage to the rabbit urinary bladder

Introduction and hypothesis  

There are a number of lower urinary tract dysfunctions (LUTD) that occur primarily in women. Our hypothesis is that cyclical estrogen will produce LUTD in part by the generation of free radicals and oxidative damage to cellular and subcellular membranes.     

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 (H₂O₂) or in vitro ischemia–reperfusion (I/R) which represents two in vitro models of oxidative damage.     

Effects of estrogen and progesterone on urinary bladder in female rabbit: evaluation by quantitative morphometric analysis

OBJECTIVES: To investigate possible effects of estrogen and/or progesterone on the histologic characteristics of female rabbit urinary bladders, we carried out quantitative morphometric analysis of the rabbit bladders. METHODS: Mature female rabbits were treated by ovariectomy with and without successive estrogen and/or progesterone administration. Area densities of the connective tissue (CT) and smooth muscle (SM) cells, the area of single SM cells, and the thickness of the bladder wall were determined by computer-assisted quantitative morphometric analysis. RESULTS: Six weeks after each treatment, ovariectomy alone resulted in a decrease in CT density of the bladder. Successive estrogen treatment increased the bladder wet weight and SM cell density within the bladder wall. Progesterone treatment reduced CT degradation in ovariectomized rabbits. Sex steroids did not significantly influence the area of each SM cell. There was no significant difference in histologic characteristics between the rabbits treated by estrogen alone and those treated by combination (estrogen and progesterone) therapy. CONCLUSIONS: Ovariectomy and successive hormonal replacement therapy resulted in morphologic changes within the rabbit urinary bladder. Cotreatment with progesterone did not significantly change the morphologic findings produced by estrogen treatment alone.     

Effect of age on the response to in vitro ischemia and reperfusion of the rabbit bladder

BACKGROUND/AIMS: Urinary bladder dysfunction secondary to outlet obstruction is a common condition, seen in both children and adults. Using in vitro models of ischemia/reperfusion (I/R), we compared the contractile and biochemical responses of bladders isolated from young and old rabbits. MATERIALS AND METHODS: Fourteen male New Zealand White rabbits were separated into two groups of 7. Group 1 (young) included rabbits 6-7 weeks old and group 2 (old) consisted of rabbits 2 years old. Isolated bladder body strips were subjected to in vitro I/R, and the effects on contractility were determined. The strips were then frozen and stored for quantitative malondialdehyde (MDA) analysis. RESULTS: Contractile responses of young and old rabbit bladders to all forms of stimulation--field stimulation (FS), carbachol and potassium chloride (KCl)--were equal. The rate of tension to 32 Hz FS was significantly higher for group 1 in comparison with group 2. The old and young rabbits were equally sensitive to I/R in regard to FS, but the old rabbits were more sensitive to I/R in regard to carbachol and KCl stimulation. Basal MDA concentration of both young and aged bladder strips were similar. Ischemia mediated a significant increase in MDA in bladder strips from both young and old rabbits, but the MDA level was significantly greater for the young than for the old. CONCLUSION: Although the level of oxidative damage was greater in the young rabbit bladders, functionally, the old rabbit bladders were more sensitive to I/R damage.     

Functional response of the rabbit urinary bladder to anoxia and ischemia

The effects of in-vitro anoxia and in-vivo ischemia on bladder function were studied using the in-vitro whole-bladder model. In addition to assessing the ability of the bladder to empty, this system provides a quantitative measurement of contractile activity. Anoxia was produced by equilibrating the isolated bladder with nitrogen instead of oxygen and ischemia was produced by clamping the arteries supplying the bladder for a period of one hour. The results of these studies can be summarized as follows: (1) The contractile response of the bladder to bethanechol did not decrease until the oxygen tension fell below 20% of normal; below this value the response decreased rapidly. (2) The ability of the bladder to contract declined gradually, as did the intracellular concentration of adenosine triphosphate. (3) The ability of the bladder to empty was lost immediately upon changing to an anoxic medium, as was the ability of bethanechol to produce a sustained contraction. (4) The bladder was able to fully recover from 60 minutes of in-vitro anoxia, whereas 60 minutes of in-vivo ischemia resulted in a permanent reduction in the contractile response to bethanechol.     

Autonomic innervation of rabbit urinary bladder following estrogen administration

The effects of estrogen administration on the autonomic innervation of the rabbit urinary bladder were studied. Immature female white rabbits were injected twice daily with estrogen (150 μg./Kg.) for four consecutive days. Control animals received injections of vehicle alone. The adrenergic innervation was identified using the glyoxalic acid method of catecholamine histofluorescence. The cholinergic innervation was visualized utilizing specific acetylcholinesterase staining. Additionally, the effect of estrogen administration on the response of smooth muscle strips of urinary bladder to specific autonomic agonists was determined. Estrogen administration induced a moderate increase in the adrenergic innervation of the rabbit bladder detrusor, whereas no change could be observed in the cholinergic innervation. It should be noted, however, that whereas the adrenergic innervation in the bladder of the control animal was sparse, the cholinergic innervation in the bladder body was quite dense. Estrogen induced a marked increase in the response to alpha -adrenergic (methoxamine) and muscarinic cholinergic (bethanechol) agonists. No alterations were noted in the response to beta-adrenergic agonists (isoproterenol). These findings indicate that the urinary bladder responds as a target organ of estrogen-induced alterations in autonomic innervation.     

Functional effects of in vitro obstruction on the rabbit urinary bladder

Bladder outlet obstruction has been the subject of numerous clinical and experimental investigations. Although these studies have demonstrated that the bladder can respond to outlet obstruction with muscular hypertrophy and hyperplasia, resulting in markedly altered morphological and functional characteristics, the nature of the direct effect of obstruction on the ability of the bladder to empty is relatively unknown. If one regards the bladder as a simple pump mechanism, decreasing the diameter of the outlet port (obstruction) would be expected to increase the work the bladder muscle would have to do in order to empty. The purpose of this present investigation is to better define the physical nature of the increased stress placed on the normal bladder via partial outlet obstruction. For these studies, the in vitro whole bladder is an appropriate model for several reasons: 1) one has complete control of initial intravesical volume, pressure, outlet diameter, and outlet resistance, and 2) this model quantitatively measures the effect of pharmacological agents and electrical stimulation on intravesical pressure and the ability of the bladder to empty. The results of these studies indicate that the obstructed bladder requires an increased pressure to empty. Although the obstructed bladder can empty completely, the rate of emptying is reduced significantly and the time required to completely empty is significantly increased. The obstructed bladder fatigues rapidly with repetitive stimulations, whereas the normal bladder is far less subject to fatigue. The physical alterations observed in these studies would play a direct role in the development of the functional alterations observed in in vivo obstruction.     

Effect of chronic nitrofurantoin on the rabbit urinary bladder

Interstitial cystitis is a pathological condition whose symptoms mimic urinary tract infection and include urgency, frequency, and moderate to severe pain. Many more women than men are affected, with antibiotic therapy being the usual first treatment approach based on symptomology. Some clinicians believe that chronic antibiotic therapy may play an etiological role in interstitial cystitis; however neither clinical nor experimental data support their opinion. The implied pathogenesis of antibiotic injury is an alteration of the bladder mucosa and its protective mucin coating to allow urine-mediated damage to the bladder wall. The purpose of this study is to evaluate rabbit urinary bladder function and morphology during chronic nitrofurantoin administration. The results demonstrate that up to twelve months of chronic nitrofurantoin administration produce no changes in 1) bacterial adherence to the rabbit bladder mucosa, 2) specific antibacterial adherence activity of the bladder mucin, and 3) ultrastructure of the mucosa, submucosa, and muscularis.     

Effect of acute complete obstruction on the rabbit urinary bladder

Studies on the effect of partial bladder outlet obstruction demonstrate that significant alterations in urinary bladder structure and function occur within 24 hours of the creation of the obstruction. These studies suggest that many of the functional and structural alterations following partial outlet obstruction may result from the initial overdistension which occurs within the first 24 hours. In these present studies, we investigated the time course of the effect of complete obstruction of the rabbit urinary bladder on the contractile response to bethanechol and field stimulation and on the muscarinic receptor density. Male White New Zealand rabbits were divided into five groups: controls, four, eight, 20, and 24 hours of complete obstruction. The results demonstrated that there was a significant decrease in both the contractile response to muscarinic stimulation and muscarinic receptor density at four hours following outlet obstruction (at a time when there was no bladder overdistension). The receptor density and contractile response to stimulation further decreased over the 24 hour period. These studies indicate that the initial decrease in muscarinic receptor density and contractile response to muscarinic stimulation may be mediated in part by the high level of spontaneous contractile activity induced by ligation of the urethra.     

Effect of urinary bladder outlet obstruction on the rabbit ureter

Ureters of mature New Zealand white male rabbits were studied at 7 and 14 days following partial bladder outlet obstruction. Muscarinic receptor density; intracellular concentrations of DNA, RNA, lipid, and hydroxyproline; and functional characteristics of the whole ureter were assessed as a means of characterizing the ureteral secondary response to partial bladder obstruction. Wet weight increased 2-fold by day 7 and almost to 3-fold by day 14. Ureteral length increased 15% by day 7 and 29% by day 14. DNA concentration varied only slightly from controls while RNA increased by 68% by day 7 but returned to near normal range by day 14. Total hydroxyproline content increased, although hydroxyproline concentration per mg tissue decreased. Total lipids increased by 73% over the first 7 days but subsequently decreased to 27% above control levels by day 14. After 1 week of obstruction, muscarinic receptor density was 35.5% of controls, and after 2 weeks of obstruction, muscarinic density was 52.5% of controls. Although muscarinic receptor density decreased significantly, the number of receptors per ureter did not substantially change. Flow studies demonstrated increased opening pressures at 7 days and decreased opening pressures by day 14. Flow as a function of constant pressure was not significantly changed by the 7th day but increased 24% by the 14th day. Bethanechol (muscarinic agonist) caused similar decreases in flow in control and 1- and 2-week obstructed ureters. These studies indicate that 1 week following partial outlet obstruction, ureters demonstrate increased protein synthesis and lipid deposition resulting in increased weight and length. By the 2nd week, protein synthesis and lipid content begin to return to normal. Weight and length remain significantly increased and the capacity for flow through the ureter improves.     

Effect of bladder outflow obstruction on the innervation of the rabbit urinary bladder.

The effects of bladder outflow obstruction on the innervation of the bladder were studied using a rabbit animal model. Partial occlusion of the bladder neck was obtained by the placement of a silk ligature at that level; control animals underwent a sham procedure. After a 3-month period, the presence of outflow tract obstruction was confirmed using urodynamic studies. The animals were then killed and pharmacological assessments of the bladder innervation undertaken. Detrusor muscle strip studies provided evidence of damage to the cholinergic innervation of the detrusor. Also, muscle strips from obstructed animals showed reduced inhibitory responses to beta-adrenergic stimulation with isoprenaline. In addition to these muscle strip studies, the bladder content of the neuropeptide substance P was assayed, but no significant change was observed in response to obstruction. This finding suggests that substance P-containing sensory nerves may be spared from the denervating effect of bladder outflow obstruction.     

The response of autonomic receptors to castration and testosterone in the urinary bladder of the rabbit

The effects of castration and testosterone on the autonomic receptor density and contractility in the urinary bladder smooth muscle of male rabbits were compared to untreated animals. Four groups of rabbits were studied over a similar time span with Group 1 animals serving as the untreated controls. Two groups (Groups 2 and 3) were castrated 28 days prior to sacrifice, Group 2 animals received corn oil for 14 days, and Group 3 animals received testosterone, 10 mg./day, for 14 days. The Group 4 animals were non-operated and received testosterone 10 mg./day for 14 days. Ligand saturation binding studies for alpha adrenoceptors in the bladder base and proximal urethra were performed with [3H]dihydroergocryptine ([3H]DHE). Muscarinic cholinergic receptors (MChR) were assayed with [3H]quinuclidinyl benzilate ([3H]QNB) and beta adrenoceptors with [125I]iodocyanopindolol ([125I]CYP) on the detrusor smooth muscle. Castrated Group 2 animals showed no significant change in receptor density with either [3H]QNB or [125I]CYP in detrusor muscle, but did exhibit a significant reduction (59%) of alpha adrenoceptors in the bladder base-urethra. The testosterone treated castrate and testosterone treated non-operated animals had significant increases in the MChR density, but no change in the alpha adrenergic, or beta adrenergic receptor density as compared to untreated controls. Cumulative dose response contractile studies were performed with carbachol on detrusor muscle strips and with phenylephrine on bladder base strips in isolated organ baths. The contractile studies on muscles from Groups 1, 2 and 3 showed no change in the ED50 or maximal contractile strength between control, castration or testosterone treated castrated animals. The ratio of wet bladder weight as compared to total body weight between each of the treatment groups showed a slight increase in both of the testosterone treatment groups. It was concluded that castration down regulates the alpha adrenergic receptors of the bladder base, while testosterone treatment increases the density of MChRs, and increases the ratio of the bladder to total body weight. Although no contractile changes were observed in the bladder base tissue it is conceivable that longer chronic testosterone deficits might ultimately affect the bladder outlet resistance in the male because of the reduced alpha adrenergic receptor density.     

The functional and structural response to distention of the rabbit whole bladder in vitro

PURPOSE: We investigated the effects of distention on intravesical pressure generation in response to stimuli in relation to its effects on bladder wall thickness and morphology. MATERIALS AND METHODS: A total of 25 New Zealand White rabbits were separated into 5 groups of 5 each. Each in vitro whole bladder preparation was filled with saline to 5% (undistended), 25%, 50%, 100% or 125% capacity (60 ml.). The effects of field stimulation, adenosine 5'-triphosphate, carbamylcholine chloride (carbachol) and KCl on intravesical pressure generation were determined. Additional bladders were similarly distended (3 at each percent of capacity), fixed in formalin and evaluated by semiquantitative morphometry. RESULTS: Pressure generation in response to field stimulation was maximal at 5% of capacity and it decreased progressively thereafter. Compared with the responses of undistended bladders, at 25% capacity the response to carbachol increased and then decreased progressively above 25%. Distention had no significant effect on the response to KCl. Interestingly pressure generation in response to adenosine 5'-triphosphate was increased more than 4-fold at 25% bladder capacity compared with 5% and it did not change significantly thereafter. Bladder wall thickness was decreased more than 50% at 25% capacity and by an additional 12% at 125% capacity. The greatest thinning was observed in the mucosa compared with the submucosa and muscularis. CONCLUSIONS: Bladder distention significantly reduced pressure generation in response to field stimulation only, indicating that distention reduced the effectiveness of synaptic transmission without altering muscarinic receptor function or membrane depolarization. The greatest change (decrease) in the response to field stimulation occurred between 5% (undistended) and 25% capacity, which was the change in distention that most affected bladder wall thinning.     

Effect of estrogen treatment on the peroxidase activity and estrogen receptors in the female rabbit urogenital tissues.

The effect of one, 4 and 8 weeks of continuous estrogenization of rabbit on peroxidase (PO) activity and on both cytoplasmic and nuclear estrogen receptors was studied in the uterus, vagina, urethra and urinary bladder. Whereas the peroxidase activity in the urogenital tissue of untreated controls was near zero, after one week of estrogen treatment it increased very substantially in the uterus and vagina and much less dramatically in the urethra or urinary bladder. With continuation of estrogen treatment for 4 or 8 weeks the PO decreased by 80-90% in the uterus and vagina and by only 40% in the urethra. After one week of estrogen treatment the density of both cytosolic and nuclear estrogen receptors decreased by several fold in both the uterus and vagina, whereas it decreased by 30-50% in the urethra and bladder. The concentration of both cytosolic and nuclear receptors decreased further although less dramatically with continuing estrogen treatment, up to 8 weeks, in all tissues. These data suggest that although the general pattern of responses to estrogen showing an initial increase in PO followed by a reduction with continuing estrogen treatment is the same in all urogenital tissues, the responses seem to have a prolonged time scale in the case of lower urinary tract tissues. The quantitative aspect of the response generally corresponds with the density of estrogen receptors in the urogenital tissues.