Biomedical subjects

Robert M Levin

Publications and source records attributed to Robert M Levin.

54 records · Page 3Linked to original sources

PubMed2003

Structural and vascular response of normal and obstructed rabbit whole bladders to distension.

OBJECTIVES: To investigate the structural and morphologic effect of distension after partial outlet obstruction in rabbits. METHODS: Thirty male New Zealand white rabbits were separated into two groups: control (sham) and partial outlet obstruction (3 weeks). Three rabbits from each group were distended to 5%, 25%, 50%, 100%, and 125% of capacity. Each bladder was fixed at the volume in buffered formalin for 6 to 8 hours. Sections of dorsal and ventral bladder were blocked, and cross sections were evaluated. Quantitative morphometry was performed, and CD31 immunohistochemistry was used to characterize the vascularity. RESULTS: Partial outlet obstruction resulted in increased bladder weight and capacity and increased thickness of the mucosa, submucosa, detrusor, and serosa. In the control bladder, the greatest thinning was seen between 5% and 25% capacity, and in the obstructed group, the greatest thinning occurred between 25% and 50%. The level of vascular collapse was significantly greater for the control bladders than for the obstructed bladders at all levels of distension. Finally, the obstructed bladders showed a significantly greater level of vascularity in the submucosa than the control bladders. CONCLUSIONS: Normal bladder distension resulted in significant morphologic changes when the bladder was distended to 25% of capacity but changed relatively little between 25% and 125%. However, distension of the obstructed bladder resulted in significant morphologic changes when the bladder was distended from 25% to 50% of capacity but changed relatively little between 50% and 125%.

Animals↗

PubMed2003

The effect of vitamin E on the response of rabbit bladder smooth muscle to hydrogen peroxide.

There is increasing evidence that ischemia, reperfusion, and the generation of free radicals are major etiological factors in the progression of bladder dysfunction after partial outlet obstruction. In vitro studies demonstrated that the magnitude of contractile dysfunction following exposure of bladder smooth muscle to hypoxia followed by re-oxygenation was related to the level of lipid peroxidation indicating that membrane lipid peroxidation participated in the contractile failure induced. Recent studies demonstrated that incubation of isolated strips of bladder smooth muscle with hydrogen peroxide (H2O2) result in progressive contractile dysfunctions and is associated with progressive increases in MDA (peroxidation product). The current study investigates if feeding rabbits a diet high in vitamin E protects the bladder from the effects of in vitro H2O2. Sixty-four male New Zealand White rabbits were separated into two groups: The rabbits in group 1 were fed a normal diet (28 rabbits) whereas the rabbits in group 2 were placed on a diet enriched with alpha-tocopherol (36 rabbits). After 3 weeks on the normal or high E diet, each rabbit was anesthetized and the bladder excised and cut into 6 isolated strips of bladder detrusor. Each strip was mounted in individual 15 ml baths containing oxygenated Tyrode's solution. The contractile responses to field stimulation (FS), carbachol. and KCl were determined. The strips were washed and exposed to one of the following concentrations of hydrogen peroxide (H2O2): 0% (control), 0.0625, 0.125, 0.25, 0.5, 1.0 and 3.0% for a period of 1 h. At the end of the hour each strip was washed free of H2O2 and a second set of contractile responses were performed and compared to the first set. At the end of the experiment, each strip was frozen and stored at -70 degrees C for analysis of malondialdehyde (MDA) as a measure of peroxidation. In both groups, H2O2 produced similar dose dependent decreases in the contractile responses to all forms of stimulation. In the normal-diet group H2O2 produced a dose dependent increase in MDA formation, whereas in the high E group there were no increases in MDA at any concentration of H2O2. Feeding rabbits a diet high in vitamin E protected the bladder smooth muscle from peroxidation, but had no significant effect on the contractile dysfunctions mediated by direct incubation with H2O2.

Animal Feed↗

PubMed2003

Response of the fetal sheep bladder to urinary diversion.

PURPOSE: In adults urinary diversion results in bladder atrophy and a rapid decrease in contractile function. Little is known about the effects of urinary diversion on bladder development. In this regard we characterized the responses of fetal sheep bladder strips obtained from animals that underwent urinary diversion. MATERIALS AND METHODS: Urinary diversion was performed on fetal sheep after 90 days of gestation (term 147 days) and bladder tissue was obtained 2 weeks later. Contractile and relaxant responses of full-thickness bladder strips were tested. RESULTS: Bladders from fetal sheep subjected to urinary diversion weighed significantly less than control fetal bladders. Histological studies demonstrated marked connective tissue infiltration and the reorganization of smooth muscle elements. Carbachol stimulated a tonic contraction, while field stimulation administered during the tonic contraction elicited a phasic relaxation or a biphasic response, consisting of an initial relaxation and then a phasic contraction in control and diverted bladders. Contractile responses of defunctionalized strips to carbachol were significantly less than those of control bladder strips. Contractile responses of defunctionalized bladder strips to field stimulation at 1 Hz. were significantly greater than those of control strips. Responses of the 2 sets of fetal bladder strips to higher frequencies were similar, as were the contractile responses to adenosine triphosphate and KCl. Field stimulated relaxations in the presence of carbachol stimulated contraction of defunctionalized bladder strips were significantly greater than those of control strips, while the relaxant responses of each set of fetal bladder strips to isoproterenol and nitroprusside were similar. CONCLUSIONS: Urinary diversion in normal fetal sheep resulted in marked structural changes, reduced carbachol stimulation and increased field stimulation relaxation.

Animals↗

PubMed2003

Acute intravesical infusion of a cobalt solution stimulates a hypoxia response, growth and angiogenesis in the rat bladder.

PURPOSE: Experimental partial bladder outlet obstruction of rats induces a bladder growth and remodeling process similar to that in humans with benign prostatic hyperplasia. Previously we have proposed that bladder hypoxia associated with partial bladder outlet obstruction is a stimulus of this bladder growth process. We report our results of testing the acute effects of a simple chemical agent (cobaltous ion) known to mimic hypoxia in the rat bladder. We measured its ability to effect bladder gene expression, angiogenesis and growth processes. MATERIALS AND METHODS: Adult rats were divided into 2 groups. One group (controls) received intravesical saline 3 times for 30 minutes in 6 days and the other received intravesical saline with 100 microM. CoCl(2) at the same times. All animals also received continuous infusion of BrdU for the 6-day period through an implanted osmotic pump. Portions of the bladders from these rats were fixed, sectioned, stained for microscopic analysis and immunohistochemically stained to identify BrdU positive cells and vascular elements via factor VIII staining. Other portions were frozen, extracted for proteins and the proteins were comparatively analyzed for the expression of hypoxia inducible factor-1alpha and vascular endothelial growth factor on Western blots. RESULTS: Bladders infused with CoCl(2) showed extensive expansion of the submucosal region, which was significant compared with that in saline infused bladders. Cells in this expanded region as well as cells within the urothelium were found to be extensively labeled with BrdU, in contrast to control bladders, which had rare BrdU labeled cells in any region. Immunohistochemical analysis for factor VIII showed that the submucosal region of cobalt treated rats contained numerous small vessels and microvessels that were not apparent in controls. These cellular changes were consistent with our finding of increased hypoxia inducible factor-1alpha and vascular endothelial growth factor protein expression in cobalt treated bladders compared with controls. CONCLUSIONS: Acute intravesical instillation of cobalt ion solution into the rat bladder initiated a hypoxia response accompanied by increased bladder angiogenesis and growth. This finding supports the idea that hypoxia is a stimulus for bladder growth subsequent to partial bladder outlet obstruction.

Administration, Intravesical↗

PubMed2003

The effect of ovariectomy and estradiol on rabbit bladder smooth muscle contraction and morphology.

PURPOSE: The bladder can be considered a target organ for the actions of estrogen. Decreases in circulating estrogen after menopause have been associated with bladder dysfunctions, including incontinence and detrusor instability. We determined the effects of estrogen on rabbit bladder contractile function and morphology. MATERIALS AND METHODS: Female New Zealand White rabbits were ovariectomized or sham operated and treated with vehicle or estradiol (1 mg/kg weekly) for 5 weeks. Serum estradiol concentration was monitored every 2 weeks. After treatment each rabbit was anesthetized, the bladder was catheterized, cystometry was performed, and the bladder was removed for contractile and morphological studies. Apoptosis in paraffin embedded rabbit bladder tissue was detected using in situ end-labeling, specifically terminal deoxynucleotidyl-transferase nick end labeling or the TUNEL assay. RESULTS: Ovariectomy resulted in a 50% decrease in circulating estrogen and estradiol treatment resulted in a 5-fold increase. Ovariectomy had no significant effects on bladder capacity, micturition pressure or bladder weight; whereas estradiol treatment resulted in significant increases in bladder capacity and bladder weight. Ovariectomy resulted in a decreased rate of tension generation in response to field stimulation, carbachol and KCl. Estradiol resulted in increased contractile responses to FS and carbachol, and an increased rate of tension generation for carbachol and KCl. Histologically ovariectomy resulted in significant urothelial apoptosis, which was not present in the sham operated or estradiol treated groups. Estradiol treatment resulted in the appearance of large cytoplasmic vacuoles in the urothelium and significant smooth muscle hypertrophy. CONCLUSIONS: These findings demonstrate that bladder function and structure can be significantly affected by modulating the circulating estrogen level. In addition, estrogen given in pharmacological doses can have a significant hypertrophic effect on bladder smooth muscle, resulting in increased contractile function.

Animals↗

PubMed2003

Effect of age on hydrogen peroxide mediated contraction damage in the male rat bladder.

PURPOSE: Recent studies introduced the concept that reactive oxygen species (ROS) may be a major factor in the progressive deterioration of bladder function induced by benign prostatic hyperplasia in men and following partial outlet obstruction in animals. We determined whether bladder contraction sensitivity to ROS changes with aging. Using H2O2 to simulate ROS damage we compared the sensitivity of the contractile responses of bladder smooth muscle isolated from young and elderly rats to H2O2. MATERIALS AND METHODS: Male 3-month-old (young) and 12-month-old (elderly) Sprague-Dawley rats were used for this study with 24 per group. Each rat was anesthetized and the bladder was excised. Two longitudinal strips were cut from the bladder body. Each strip was placed in individual 15 ml baths containing oxygenated Tyrode's solution at 37C. Each strip was stimulated at 32 Hz for 20 seconds with pulses 1 millisecond in duration at 80 V. After electrical field stimulation (EFS) the response to 20 microM carbachol and 120 mM KCl was determined. The bath solution was then exchanged for H2O2 solutions of 6 different concentrations including 0%, 0.0625%, 0.125%, 0.25%, 0.5% and 1.0%. After 1 hour of exposure to H2O2 the tissues were washed free of H2O2, and the response to EFS, carbachol and KCl were measured again. At the end of the experiment the strips were weighed and frozen at -70C for malondialdehyde analysis. RESULTS: The magnitude of the contractile responses of the young and elderly rats to all forms of stimulation were equal. Hydrogen peroxide caused a dose dependent decrease in the contractile responses of bladder strips to all forms of stimulation. Contractile responses to carbachol and KCl were more sensitive to H2O2 than to EFS. Contractile responses of bladder strips isolated from elderly rats were significantly more sensitive to H2O2 damage than strips isolated from young rats. Malondialdehyde generation of bladder strips isolated from elderly rats was significantly greater than those from young rats. CONCLUSIONS: It is suggested that aging increases the sensitivity of detrusor contraction to oxidative damage.

Age Factors↗

PubMed2003

Reperfusion injury of the rat bladder is worse than ischemia.

PURPOSE: Previous studies have demonstrated that in vivo and in vitro ischemia of the bladder results in decreased contractile responses. However, to our knowledge the effect of reperfusion following ischemia of the bladder is not known. MATERIALS AND METHODS: Adult male rats were subjected to bilateral bladder ischemia and varying periods of reperfusion. In vivo ischemia was created for 4 hours by reversibly clamping the 2 vesical arteries for 4 hours. Reperfusion was produced by removing the clamps and allowing the animals to recover for 1 day, 1 week or 1 month after surgery. Following recovery bladders strips were studied using field stimulation (FS), carbachol and KCl. The maximal contractile response and rate of response generated were recorded digitally and analyzed. RESULTS: The maximal responses to FS, carbachol and adenosine triphosphate (ATP) were not decreased by 4-hour ischemia alone, whereas the response to KCl was decreased significantly. The contractile responses to FS and KCl were significantly decreased after 1 day and 1 week of reperfusion. Responses after 1 month of reperfusion were increased significantly compared with responses after 1 week of reperfusion. The responses to ATP were not affected by ischemia or reperfusion. The contractile response to KCl was significantly more sensitive to ischemia than the responses to carbachol, ATP or FS, whereas the contractile response to FS was significantly more sensitive to reperfusion than the other forms of stimulation. CONCLUSIONS: This study demonstrates clearly that injury by reperfusion following ischemia is more detrimental than the effects of ischemia alone and FS contraction is the most sensitive form of stimulation to reperfusion damage. This study also demonstrates the ability of the bladder to recover from ischemic and reperfusion injuries.

Adaptation, Physiological↗

PubMed2003

Excitatory alpha1-adrenergic receptors predominate over inhibitory beta-receptors in rabbit dorsal detrusor.

PURPOSE: We mapped the regional distribution of alpha1 and beta-adrenergic receptors (ARs) in rabbit ventral and dorsal bladder, and characterized the alpha1-AR subtypes responsible for norepinephrine induced contraction of rabbit dorsal detrusor smooth muscle. MATERIALS AND METHODS: Responses to norepinephrine in the absence and presence of adrenergic antagonists were measured in strips taken from clearly defined regions of male rabbit ventral and dorsal bladder. RESULTS: In the absence of antagonists ventral strips from the bladder body relaxed in response to norepinephrine, while those from the ventral base contracted. Dorsal strips from the bladder dome also relaxed in response to norepinephrine but dorsal strips from the mid and lower body, and the base contracted. All contractile responses were antagonized by incubation with prazosin. Characterization of the alpha-AR subtypes present in dorsal strips from the mid and lower bladder body using BMY 7378 (8-[2-[4 to 2(-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5] decane-7,9-dione dihydrochloride), chloroethylclonidine, 5-methylurapidil and WB 4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane) suggested that the alpha1-AR subtype responsible for the contraction is the alpha1A or alpha1L-AR. CONCLUSIONS: The male rabbit bladder contains at least 4 heterogeneous regions with differing functional responses to adrenergic stimulation, that is 1) the dorsal and ventral dome, where beta-ARs predominate, 2) the ventral detrusor, where beta-ARs predominate, 3) the dorsal detrusor, where alpha1-ARs predominate, and 4) the dorsal and ventral bladder neck, where alpha1-ARs predominate.

Adrenergic alpha-Agonists↗

PubMed2003

Effect of DHLA on response of isolated rat urinary bladder to repetitive field stimulation.

Lipoic acid is an essential coenzyme in the oxidation of pyruvate and alpha-ketoglutarate. It is easily converted to its reduced form, dihydrolipoic acid (DHLA), in vivo thereby forming a redox pair. DHLA is important in the maintenance and integrity of specific neuronal and subcellular membranes. In the present study we investigated the effect of DHLA on the response of isolated rat bladder strips to repetitive field stimulation (FS), a method used to exhaust synaptic stores of acetylcholine resulting in nerve and synaptic damage. Isolated strips of rat urinary bladders were separated into 4 groups. Group 1 strips were incubated with choline + acetyl-CoA; Group 2 strips with choline, acetyl-CoA + DHLA; and Group 3 with DHLA. Group 4 strips were controls. All strips in Groups 1-3 were subjected to 2 h of repetitive FS followed by 2 h of recovery. DHLA had no effect on the progressive decrease in contractile response observed during repetitive stimulation. However, strips incubated in the presence of DHLA showed a significantly greater degree of recovery than strips incubated in the absence of DHLA. We believe that the protection of the contractile response is related to DHLA's ability to protect nerve and/or muscle membranes from oxidative damage.

8,11,14-Eicosatrienoic Acid↗

PubMed2002

Cloning of COX4 cDNA from the NZ white rabbit and expression of this gene in bladder smooth muscle following partial outlet obstruction.

We cloned and characterized the rabbit COX4 coding sequence, then examined expression of the gene in bladder smooth muscle following partial obstruction of the bladder outlet. Obstruction was done surgically in male NZ white rabbits and bladders procured at 1, 3, 5, 7, and 14 days post-obstruction (two animals/time). RNA, then cDNA, was prepared from smooth muscle from each sample. Cloning was done from cDNA by PCR. COX4 mRNA in smooth muscle was defined by real-time RT-PCR, using cDNA from bladder samples. The rabbit COX4 coding sequence specifies a protein of 169 amino acids, including a membrane transit sequence for mitochondrial (mt) entry. Expression of COX4 in bladder smooth muscle decreased by 80% in the first day post-obstruction and remained low through 14 days post-obstruction. Cytochrome oxidase activity decreased sharply in the first 3 days post-obstruction in smooth muscle, in concert with COX4 transcript levels. Partial outlet obstruction of the rabbit urinary bladder results in attenuation in smooth muscle of mRNA synthesis from the COX4 gene. In turn, this results in attenuated activity of mt electron transport due to limited availability of nucleus-encoded components required for the assembly of cytochrome oxidase.

Animals↗

PubMed2002

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.

Adenosine Triphosphate↗

PubMed2002

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.

Adenosine Triphosphate↗

PubMed2002

Activation of choline acetyl transferase by dihydrolipoic acid.

In a previous article we present results that demonstrated that the enzyme, choline acetyl transferase, was strongly activated by dihydrolipoic acid and that the oxidized form of this substance, lipoic acid itself, was an inhibitor of the enzyme and overcame the stimulatory effects of reduced lipoic acid. The experiments presented in this article show that dialysis of a partially purified preparation of choline acetyl transferase causes complete disappearance of enzyme activity and that addition of dihydrolipoic acid restores activity towards normal. In addition we present experiments with extracts of rat brain and heart as well as rabbit bladder tissue. In these extracts dihydrolipoic acid strongly activates the enzyme. Dialysis of brain and heart extracts causes loss of activity with partial restoration of activity by addition of dihydrolipoic acid. Reduced glutathione has no ability to stimulate activity of the enzyme. We conclude that the results of these experiments strongly support the view that dihydrolipoic acid acts as a coenzyme in the choline acetyl transferase reaction.

Animals↗

PubMed2002

Human immunodeficiency virus infection and the thyroid.

Abnormalities of thyroid function are associated with a number of systemic conditions, including patients infected with human immunodeficiency virus (HIV). Most patients with early HIV infection and a stable body weight have normal thyroid function. Subtle abnormalities of a number of thyroid function tests have been reported during the early asymptomatic phase of HIV disease. These include an inappropriately normal triiodothyronine (T(3)) and reduced reverse triiodothyronine (rT(3)), and increased thyroxine-binding globulin (TBG) levels. Opportunistic infections involving the thyroid gland, neoplasms such as lymphoma and Kaposi's sarcoma, and medications can alter the thyroid function in individuals with more advanced HIV infection. If thyroid dysfunction is diagnosed in an HIV-infected patient, it should be treated in the usual manner. However, high index of suspicion and caution in the interpretation of thyroid function tests in patients with HIV disease are needed for optimal diagnosis and treatment.

Anti-HIV Agents↗

PubMed2002

Hypoxia and an angiogenic response in the partially obstructed rat bladder.

Previous molecular and blood flow studies performed on animal models of partial bladder outlet obstruction (PBOO) caused us to propose that bladder hypoxia/ischemia was a significant effector of the cellular and functional changes that occur in the bladder as a result of this condition. To confirm the occurrence of hypoxia in the partially obstructed bladder, we obtained rat bladders at increasing intervals following PBOO and measured biomarkers of hypoxia (intracellular formation of hypoxyprobe-1 adducts and expression of hypoxia inducible factor-1 alpha [HIF-1 alpha] protein) and whether such hypoxia might elicit an angiogenic response in the tissue. Rats receiving PBOO or controls were treated with hypoxyprobe-1 at increasing intervals subsequent to surgery and their bladders were sectioned and immunostained using an antibody that detects hypoxyprobe-1 adducts. Control rat bladders were unstained, whereas intense, but regionally restricted, hypoxyprobe-1 immunostaining was detected in all obstructed bladders in a unique pattern that changed over time. Proteins were extracted from bladders removed from similarly treated rats and were analyzed for the expression of the HIF-1 alpha protein as well as for expression of angiogenic regulatory factors (vascular endothelial growth factor, angiopoietin-1, and endostatin) using Western blotting techniques. HIF-1 alpha protein was not expressed in control bladders, however, the protein was highly up-regulated over the 2-week period after PBOO. Likewise, the expression of vascular endothelial growth factor (a downstream target of HIF-1 alpha action) and angiopoietin-1 was also up-regulated in obstructed bladders confirming an angiogenic response to this hypoxia. Enigmatically, however, expression of the antiangiogenic molecule endostatin was also up-regulated by chronic PBOO. These results further support the concept that hypoxia is involved in the cellular remodeling as well as in the progressive functional impairment exhibited by the urinary bladder after PBOO.

Angiopoietin-1↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗