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

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Effects of chronic partial outlet obstruction on blood flow and oxygenation of the rat bladder

PURPOSE: Experimental partial bladder outlet obstruction in rats and rabbits drives the bladder through 3 sequential responses, referred to as hypertrophy, compensation and decompensation. The hypertrophy phase, which is a period of rapid bladder growth, has previously been shown to be accompanied by a significant increase in bladder blood flow in rats and rabbits in a manner that likely supports the bladder cell growth process. However, chronic periods of obstruction in the rabbit have been shown to reduce significantly bladder blood flow, especially to the detrusor smooth muscle, corresponding with a loss of bladder contractile function or decompensation in these animals. We determined the effects of chronic 1 to 4-week partial outlet obstruction on rat bladder blood flow and directly correlated them with hypoxia in the rat bladder. MATERIALS AND METHODS: Rats underwent surgical partial bladder outlet obstruction under anesthesia. At weekly intervals after surgery relative blood flow to the bladder and spleen was measured by a fluorescent microsphere infusion technique. Sham operated rats were also studied 2 and 4 weeks following surgery. In a second experiment groups of similarly obstructed rats were treated with Hypoxyprobe-1 (Natural Pharmacia International, Inc., Research Triangle Park, North Carolina), a chemical probe for hypoxia, 3 days, 1 and 2 weeks after partial bladder outlet obstruction. The bladders were subsequently fixed and immunostained using a monoclonal antibody that detects Hypoxyprobe-1 adducts that are selectively formed in hypoxic cells. RESULTS: Neither bladder weight nor bladder relative blood flow was affected by sham surgery. Likewise, control and sham obstructed rat bladders were found to be free of Hypoxyprobe-1 reactive areas. In contrast, obstructed rats had significantly increased bladder weight at all time points. Relative weight of the obstructed rat bladders indicates the response to mild-moderate obstruction. Bladder relative blood flow in obstructed rats was significantly elevated 1 and 2 weeks after partial bladder outlet obstruction but it returned to almost control levels by 3 and 4 weeks. Hypoxyprobe-1 staining demonstrated a sequential transition of hypoxia from bladder mucosa and submucosal regions at 3 days to muscularis and serosal fibroblasts 1 week and finally to smooth muscle cells by 2 weeks after obstruction. CONCLUSIONS: In contrast to the rabbit model, global blood flow in the mild-moderate chronically obstructed rat bladder was found to be higher or nearly equivalent to blood flow in unobstructed control rat bladders. However, even in the presence of normal or above normal blood flow focal regions of hypoxia were still observed in obstructed rat bladders and these regions changed with time. These results provide a reason to understand better why rats are more resistant to the onset of bladder decompensation than rabbits and support the concept that hypoxia is involved in bladder remodeling as well as in progressive functional impairment of the bladder after partial bladder outlet obstruction.     

Novel biomarkers of bladder decompensation after partial bladder obstruction

AIMS: Partial bladder outlet obstruction (PBOO) results in marked contractile, biochemical, and histological alterations in the bladder. Our aim was to determine the time course of progressive PBOO in the rabbit and to find parameters that marked the shift to decompensation. MATERIALS AND METHODS: Twenty-four rabbits were subjected to 1, 2, 4, and 8 weeks of PBOO. Sham operated rabbits served as controls. At each time period, cystometry was performed and individual bladder strips were used for contractility studies. Full-thickness sections of bladder body from each rabbit were fixed in formalin and used to determine the vascular density and nerve density. The balance of the bladder body was separated between muscle and mucosa and was analyzed for superoxide dismutase (SOD) and catalase (CAT) activities. RESULTS: Bladder weight increased progressively and all contractile responses were reduced significantly over the course of obstruction. Markedly increased bladder weight and very large bladder volumes indicated decompensation. Nerve density was marked decreased in decompensated bladders. Similarly, SOD activity in muscle decreased progressively and was markedly lower in decompensated bladders. Although CAT activity of the muscle increased after 2-4 weeks of obstruction, it decreased markedly in decompensated bladders. CONCLUSION: This study shows that prolonged PBOO causes progressive deterioration in the rabbit bladder with decompensation after 8 weeks. Markedly decreased nerve density and severely reduced SOD and CAT activities are associated with the shift from compensated to decompensated function of the bladder. They may be excellent biomarkers of decompensation.     

Lipid signaling changes in smooth muscle remodeling associated with partial urinary bladder outlet obstruction

AIMS: Hypertrophy of the urinary bladder smooth muscle (detrusor) is associated with partial bladder outlet obstruction (PBOO). Hypertrophied detrusor smooth muscle (DSM) reveals altered contractile characteristics. In this study, we analyzed the lipid-dependent signaling system that includes phospholipase A2 in PBOO-induced DSM remodeling and hypertrophy to determine whether the release of arachidonic acid (AA) from phospholipid is altered in the detrusor. METHODS: Partial bladder outlet obstruction (PBOO) was produced by partial ligation of the urethra in New Zealand white rabbits. Two weeks after the surgery, the bladder function was studied by keeping the rabbits in metabolic cages for 24 hr. Bladders were removed from rabbits that had bladder dysfunction (increased urinary frequency and decreased void volume) and the DSM separated from mucosa and serosa. The isolated smooth muscle was incubated with [3H] AA to equilibrate the cytoplasmic AA. The level of AA release was compared with the level obtained with 2-week sham-operated rabbits. RESULTS: The rate of AA release was high in DSM from bladders with PBOO-induced hypertrophy. Carbachol stimulated AA release in control DSM but DSM from obstructed rabbits revealed no further increase from the elevated basal AA release. The half-maximal concentration of carbachol that was required to stimulate AA release from control samples of detrusor was 35 microM. CONCLUSIONS: The increased levels of AA release that are observed in this tissue after PBOO indicate the activation of phospholipase A2. The finding that carbachol could induce contraction, but not an increase in AA, indicates that the carbachol-induced contraction in the obstructed bladders is independent of lipid signaling pathways that involve AA. It is possible that the increased rate of arachidonic acid release from obstructed bladders correlates with the enhanced rates of prostaglandin production reported by other investigators from the same tissue.     

Rabbit urinary bladder blood flow changes during the initial stage of partial outlet obstruction

PURPOSE: The rabbit urinary bladder's early response to partial outlet obstruction includes bladder wall remodeling with marked urothelial and fibroblast hyperplasia (1 day) and smooth muscle hypertrophy (3-5 days) resulting in a 4-5 fold increase in bladder mass within 7 days. In this study, we examined the effect of partial outlet obstruction on bladder blood flow during the initial period of rapid growth (1-7 days). MATERIALS AND METHODS: Each New Zealand White rabbit was partially obstructed by tying a 2-0 silk ligature loosely around the vesical outlet. After 0 (unoperated), 4 hours, 1, 3, or 7 days of obstruction, 5 rabbits per group were anesthetized and the carotid and femoral arteries cannulated with polyethylene tubing. Additional rabbits receiving sham surgeries were treated like obstructed animals at 4 hours and 1 day post-obstruction (5/group). Using standard methods, fluorescent microspheres were infused through the right carotid artery. Bladder and right kidney were rapidly removed upon completion of sphere infusion; bladder mucosa and muscle were separated. Sphere densities in detrusor, mucosa, and kidney were measured by Interactive Medical Technologies, Ltd. A section of each detrusor tissue was fixed in formalin and immunostained for smooth muscle alpha-actin. RESULTS: Mucosal blood flow (0.20 +/- 0.03 ml./min./gm.) was approximately 4-fold greater than that of detrusor (0.05 +/- 0.01 ml./min./gm.). Sham surgery caused a significant increase in bladder blood flow at 4 hours post-obstruction that returned to control levels by 1 day. Both mucosal and muscle blood flows were slightly higher in rabbit bladders obstructed for 4 hours than in sham-operated rabbits, and substantially greater in those obstructed for 1 day: 0.68 +/- 0.13 ml./min./gm. (mucosa) and 0.26 +/- 0.04 ml./min./gm. (muscle). Blood flows returned to control values by 3 days post-obstruction and remained constant through 7 days. Kidney blood flow was unchanged. Although bladder weight increased 4-fold after 7 days of obstruction, the volume fraction of smooth muscle (transverse section) remained constant at approximately 40%. CONCLUSIONS: Blood flow was approximately 4-fold greater in bladder mucosa than in muscle, which may relate to the significantly higher metabolic rate and lower high energy phosphate concentration of mucosa than muscle. Partial outlet obstruction resulted in a significant increase in blood flow at 1 day post-obstruction, which coincides temporally with the early cellular hyperplasia and hypertrophy of obstructed rabbit bladder. This increase in blood flow may be an essential factor for the initial increase in bladder mass. By three days, the blood flow per gram of tissue returned to control levels. The mechanisms relating to the changes in blood flow induced by partial outlet obstruction are currently under investigation.     

Effect of oral Tadenan treatment on rabbit bladder structure and function after partial outlet obstruction

PURPOSE: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Effect of partial outflow obstruction on the distribution of free fatty acids and phospholipids in the rabbit bladder

The urinary bladder is separated into two distinct components, the mucosal epithelium (urothelium) and the underlying detrusor smooth muscle. Specific bladder dysfunctions such as partial outlet obstruction may contribute to the breakdown and damage of cell membranes. The major component of cell membranes is phospholipids, and the release of free fatty acids (FFA) from membrane phospholipids is suggestive of degradative lipase activity. The current investigation is concerned with the effect of partial outlet obstruction on the subcellular distribution of free fatty acids and phospholipids (PL) in rabbit bladder muscle and mucosa. Partial outlet obstructions were surgically created in mature male New Zealand White rabbits by standard methodology. At 2 weeks following surgery, rabbits were euthanized and the bladders, removed and separated into smooth muscle and mucosa. Muscle and mucosa were homogenized and separated by differential centrifugation to obtain separate subcellular fractions including plasma membranes, mitochondria, microsomes, and cytosol. The homogenate and supernatant fraction, free of membranes, were also saved. The free-fatty-acid (FFA) and choline-containing phospholipid (PL) content and the rate of generation of FFA were quantitated using in vitro enzymatic colorimetric methods. Relative to controls there was a significant increase in the FFA content of the obstructed smooth muscle and an increase in the PL content of the obstructed mucosa. There was an increase in FFA content in the mitochondrial fraction and a decrease in the supernatant of the obstructed smooth muscle. The PL content was reduced in the obstructed smooth muscle microsomal and supernatant fractions and was increased in the supernatant fraction of the mucosa. Endogenous lipase activity among control bladders was more than 10-fold greater in mucosa than in muscle. The FFA generation of the smooth muscle was significantly reduced by partial outlet obstruction. In conclusion, partial outlet obstruction causes bladder dysfunction due to activation of enzymes that hydrolyze cellular and subcellular membranes. The increase in endogenous lipase activity and generation of FFA among obstructed bladders indicates that the pathological state affects the membrane structure needed for normal bladder function.     

Protective effect of vitamin E on the response of the rabbit bladder to partial outlet obstruction.

PURPOSE: There is increasing evidence that ischemia/reperfusion is a major etiological factor in the progression of bladder dysfunction after partial outlet obstruction. If this evidence is correct, treatment with an antioxidant should be beneficial in rabbits subjected to partial outlet obstruction. We designed the current study to determine if diets high in alpha-tocopherol protected the rabbit bladder against dysfunction induced by partial outlet obstruction. MATERIALS AND METHODS: A total of 32 rabbits were separated into 4 groups of 8. Groups 1 and 2 were placed on a diet enriched with 1,000 IU/kg. alpha-tocopherol, and groups 3 and 4 were fed a regular diet containing 44 IU/kg. alpha-tocopherol. After 4 weeks partial outlet obstruction was created in groups 1 and 3, while groups 2 and 4 underwent sham operation. After 4 weeks of obstruction the rabbits were anesthetized and the bladders were rapidly excised. Four longitudinal strips obtained from the bladder body were used for contractility studies. The balance of the bladder body was separated between muscle and mucosa. Each section was frozen and stored at -70C for analysis of malondialdehyde as a measure of peroxidation and for alpha-tocopherol concentrations. RESULTS: Feeding rabbits a diet high in alpha-tocopherol resulted in significant protection against the development of contractile dysfunction after partial outlet obstruction. The protective effect of alpha-tocopherol was related to significantly decreased malondialdehyde and significantly increased tissue concentrations of alpha-tocopherol. CONCLUSIONS: These data indicate that a major etiology of bladder dysfunction secondary to partial outlet obstruction is related to free radical generation and resultant membrane lipid peroxidation.     

Contractile protein expression in bladder smooth muscle is a marker of phenotypic modulation after outlet obstruction in the rabbit model

PURPOSE: We determined changes in contractile protein expression before and after the relief of partial bladder outlet obstruction in the rabbit model and assessed their potential role as predictors of recovery. MATERIALS AND METHODS: We examined the ratio of the smooth muscle myosin heavy chain isoforms SM2-to-SM1, caldesmon isoform expression and bladder function in obstructed and unobstructed adult rabbit bladders. Cystometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were done to determine changes in bladder function and contractile protein expression. RESULTS: Overall we observed significant correlation of bladder weight with the SM2-to-SM1 ratio (p <0.05). Regardless of the duration of obstruction (up to 10 weeks) the ratio appeared to stabilize around a value comparable to that in fetal rabbit smooth muscle cells, suggesting a reversal of SM2 and SM1 expression to a level similar to that at the fetal stage. The pattern of h and l-caldesmon isoform expression showed an increase in l-caldesmon expression in obstructed bladders. Except for decreased leak point pressure in the obstructed group we noted no statistically significant urodynamic changes in bladder capacity or compliance. CONCLUSIONS: There is significant correlation of bladder weight, which is the best known marker of obstruction, with the SM2-to-SM1 ratio. The myosin heavy chain isoform expression ratio appears to be an indicator of phenotypic modulation in bladder smooth muscle before and after the relief of bladder outlet obstruction. Thus, it may be useful as a marker of bladder dysfunction and predictor of functional recovery. Regression to a fetal pattern of protein expression may suggest irreversible damage to smooth muscle cells, possibly limiting recovery.     

Alterations in purinergic and cholinergic components of contractile responses of isolated detrusor contraction in a rat model of partial bladder outlet obstruction

OBJECTIVE: To study the effect of 3 weeks of partial bladder outlet obstruction (BOO), compared to a sham operation, on the cholinergic and purinergic components of detrusor contractile responses to agonists and to electrical field stimulation (EFS); the expression of P2X receptor subtypes was also examined. MATERIALS AND METHODS: Partial BOO was induced in female Sprague-Dawley rats by surgically applying a jeweller's silver 'jump' ring around the urethra, such that the urethra was constricted but not closed. Sham-operated female rats underwent an identical procedure without placement of a ring. RESULTS: After 3 weeks of partial BOO the rat bladders became significantly hypertrophied, doubling in weight. Spontaneous activity was markedly increased, but the contractile response to a single bolus of KCl (120 mM) was unaltered. The neurogenic-induced contractile responses of strips of detrusor from obstructed bladders were significantly greater than those from sham-operated bladders, and the responses of strips of detrusor from obstructed bladders to EFS showed a significantly greater atropine-sensitive component than sham-operated detrusor. However, the response of detrusor strips to EFS that was susceptible to desensitization by alpha,beta-methylene ATP was not significantly changed in obstructed bladders. The sensitivity of the strips from obstructed bladders to carbachol, ATP and beta,gamma-methylene ATP was less than in sham-operated detrusor. Immunohistochemical studies showed no difference in the P2X receptor subtypes expressed on detrusor smooth muscle from obstructed and sham-operated rats. CONCLUSION: In the rat, after moderate bladder hypertrophy, the atropine-sensitive component was significantly up-regulated, but the ATP-sensitive component was marginally reduced, although not significantly. These results suggest that up-regulation of the P2X component of bladder contraction seen in humans with bladder instability, and in other species models of BOO, is not mirrored in the rat, or occurs later in the pathological process of bladder hypertrophy.     

Properties of Ca2+-Mg2+ ATP-ase in rabbit bladder muscle and mucosa: Effect of urinary outlet obstruction

The contractile response of the smooth muscle of the urinary bladder is dependent upon both the entrance of extracellular calcium through receptor-operated calcium channels and the stimulated release of calcium from the sarcoplasmic reticulum. In addition, partial outlet obstruction induces marked alterations in the utilization of intracellular calcium. Although calcium ATP-ase provides the energy for the translocation of intracellular free calcium into storage sites within the sarcoplasmic reticulum, very little is known about the properties of this enzyme in bladder muscle and mucosa. As an initial study, divalent ion specific ATP-ase activity was measured in extracts of rabbit bladder muscle and mucosa from control animals and from rabbits following partial urinary outlet obstruction. In both normal bladder muscle and mucosa, magnesium and calcium ions were equally effective in activating the enzyme. Seven days following partial urinary outlet obstruction, the ATP-ase activity in both bladder muscle and mucosa was significantly depressed by over 70%. The degree of the decreased enzyme activities observed within the muscularis and mucosa would indicate that specific membrane functions supported by divalent-ion-ATP-ase are dysfunctional. This hypothesis is supported by marked alterations in the utilization of intracellular calcium following partial outlet obstruction and the marked dysfunctions in both mucosal permeability and bacterial adherence to mucosa observed following partial outlet obstruction. © 1996 Wiley-Liss, Inc.     

Effect of doxazosin on rat urinary bladder function after partial outlet obstruction

Hypoxia induced by partial outlet obstruction is believed to play a major role in both the hypertrophic and degenerative effects of partial outlet obstruction. Doxazosin (dox) is a clinically effective alpha-adrenergic antagonist used in the treatment of symptomatic benign prostatic hyperplasia (BPH). Although the major therapeutic effect of the agent is believed to occur on the smooth muscle components of the prostate by reducing prostatic urethral resistance and thus improving emptying, dox may have part of its clinical action via effects mediated by other actions, including via spinal alpha-adrenergic receptors or direct effects on the bladder, possibly via inhibition of vascular alpha receptors. The specific aim of the current study was to determine whether dox pretreatment on rats affects blood flow to the bladder and reduces the level of bladder dysfunction induced by partial outlet obstruction. In part 1, eight rats were separated into two groups of four rats each. Group 1 received oral administration of dox (30 mg/kg) for 4 weeks; group 2 received vehicle (5% dimethyl sulfoxide). After 4 weeks of treatment, blood flow studies were performed using fluorescent microspheres and the bladders excised, frozen, and submitted to Interactive Medical Technologies (IMT) for blood flow analysis. In part 2, 32 adult male rats were separated into four groups of eight rats each. Groups 1 and 2 received oral administration of dox (30 mg/kg) for 4 weeks, groups 3 and 4 received vehicle (5% dimethyl sulfoxide). At 4 weeks, the rats in groups 1 and 3 received partial outlet obstructions and treatment continued for an additional 2 weeks. After 6 weeks of treatment (total), each rat was anesthetized, the bladder excised, weighed, and isolated strips mounted and contractility studies performed. 1) Four weeks pretreatment of rats with dox increased blood flow to the bladder in both the control and obstructed groups. 2) Partial outlet obstruction induced a mild decrease in blood flow. 3) The magnitude of the increased bladder weight in the vehicle-treated obstructed group was significantly greater than in the dox-treated obstructed group. 4) Partial outlet obstruction resulted in significant decreases in the contractile response to field stimulation in both treated and non-treated rats. The magnitude of the decreased response was significantly greater in the non-treated rats. 5) The response to potassium chloride was significantly reduced by partial outlet obstruction in the vehicle-treated group but not in the dox-treated group. 6) The time to maximal tension was significantly increased in response to carbachol, adenosine triphosphate, and potassium chloride. However, the magnitude of the increase was significantly greater for the vehicle-treated obstructed groups stimulated by potassium chloride than for the dox-treated groups. Dox treatment of rats increased blood flow to the bladder and reduced the severity of the response to partial outlet obstruction. These beneficial effects would be due to pharmacological effects on alpha-adrenergic systems outside those present in the prostate. These include effects on blood flow to the bladder, effects on the micturition centers of the central nervous system, spinal reflexes, and alpha-adrenergic receptors in the urethra and bladder.     

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.     

The effect of bladder outlet obstruction on tissue oxygen tension and blood flow in the pig bladder

OBJECTIVE: To investigate the effect of partial bladder outlet obstruction on detrusor blood flow and oxygen tension (PdetO2) in female pigs. MATERIALS AND METHODS: Detrusor-layer oxygen tension and blood flow were measured using oxygen-sensitive electrode and radiolabelled microsphere techniques in five female Large White pigs with a partial urethral obstruction and in five sham-operated controls. The effects of chronic outlet obstruction on bladder weight, and cholinergic nerve density and distribution, are also described. RESULTS: In the obstructed bladders, blood flow and oxygen tension were, respectively, 54.9% and 74.3% of control values at low bladder volume, and 47.5% and 42.5% at cystometric capacity. Detrusor blood flow declined by 27.8% and 37.5% in the control and obstructed bladders, respectively, as a result of bladder filling, whilst PdetO2 did not decrease in the controls, but fell by 42.7% in the obstructed bladders. Bladder weight increased whilst cholinergic nerve density decreased in the obstructed animals. CONCLUSION: In pigs with chronic bladder outlet obstruction, blood flow and oxygen tension in the detrusor layer were lower than in control animals. In addition, increasing detrusor pressure during filling caused significantly greater decreases in blood flow and oxygen tension in the obstructed than in the control bladders.     

Correlation of in vivo bladder blood flow measurements with tissue hypoxia

Aims

Obstructive bladder dysfunction is in part due to reduced blood flow and the resulting ischemia of the bladder smooth muscle and mucosa. Our aim was to determine if the severity and localization of ischemia could be determined by measuring blood flow to the bladder with a non-invasive probe placed on the surface of the urothelium.

Materials and methods

Twenty-four adult male rabbits (5 months, 3.5?C4.0?kg) were divided into three groups: 1??controls; 2??2?h of bilateral ischemia; and 3??partial outlet obstruction, and were evaluated after 2 weeks. Each rabbit received an intraperitoneal injection of Hypoxyprobe-1. In vivo real-time monitoring of blood flow was measured at five sites within the bladders with a laser Doppler flowmeter.

Results

For all groups, the blood flow readings showed no significant differences among the five sites. The ischemic bladders showed significant decreases in blood flow. The obstructed bladders had significantly lower blood flow than the ischemic bladders. The hypoxyprobe studies demonstrated that there was no hypoxia present in the control bladders; the mucosa of the ischemic bladders showed even hypoxia at an intermediate concentration; the obstructed bladders showed dense but even staining.

Conclusion

We have demonstrated that we can determine the severity of ischemia by surface measurement of blood flow.     

Effect of age on rabbit bladder function and structure following partial outlet obstruction

PURPOSE: We determined whether young and old rabbits respond differently to partial bladder outlet obstruction. MATERIALS AND METHODS: A total of 16 male New Zealand White rabbits were separated into 2 groups of 8 each. Group 1 consisted of young rabbits (age 7 weeks) and group 2 consisted of old rabbits (age 2 years). Four rabbits per group underwent partial outlet obstructions and 4 underwent sham operation. Four weeks following surgery individual bladder strips were used for contractile studies and the remaining tissue was examined histologically. RESULTS: Contractile responses to all forms of stimulation between the young and old sham operated groups were similar. Contractile responses to all forms of stimulation were significantly decreased to the same degree in the 2 obstructed groups. However, the rate of tension generation to field stimulation was decreased to a significantly greater degree in young vs old bladders. Although young and old bladders showed smooth muscle hypertrophy, older rabbits showed significantly greater thickening of the serosa than young rabbits. Young rabbits showed significant inflammation, hemorrhage and expansion of the lamina propria, whereas old rabbits showed none of these characteristics. CONCLUSIONS: Although there were only minor differences in the physiological response of young and old bladders to obstruction, young rabbits showed a significantly greater degree of histological damage. This may have been due to the thinner wall and greater sensitivity to distention.     

Effect of partial bladder outlet obstruction on nitrotyrosine levels and their correlation with contractile function

AIMS: It has been demonstrated that partial bladder outlet obstruction (PBOO) causes free radical generation that, in turn, results in cellular and subcellular damage. We tested the hypothesis that nitration of proteins is associated with contractile dysfunctions in obstructive bladder disease. METHODS: Thirty rabbits were subjected to 1-28 days of partial outlet obstruction. Sham operated rabbits served as controls. Western blotting was used to determine the amount of nitrotyrosine level at the protein level. At each time point, isolated strips of bladder body were mounted in individual baths and the contractile response to field stimulation (FS), carbachol, and KCl determined. RESULTS: Bladder weight increased rapidly during the first 7 days and then increased slowly thereafter. There was a fourfold increase in the amount of nitrotyrosine in the 7 day obstructed groups when compared to sham controls and the levels remain elevated at 14 and 28 days of obstruction. Contractile dysfunction in response to FS (8 and 32 Hz) was noted as early as 1 day after obstruction and increased progressively over the study period. The decrease in response to carbachol and KCl was significant only after 3 days of obstruction and the progressive increase in dysfunction was slower than with FS. CONCLUSIONS: PBOO is accompanied by an increase in nitrotyrosine, a marker of free radical damage. Simultaneously there was a progressive decrease in contractility of detrusor smooth muscles (DSMs). Nitrotyrosine may be usable as a marker of free radical damage and reperfusion injury.