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Obstructive Bladder Dysfunction: Morphological, Biochemical and Molecular Changes
European Urology Supplements
European Urology Supplements 1 (2002) 14±20
Obstructive Bladder Dysfunction: Morphological, Biochemical and Molecular Changes Robert M. Levina,*, Paul Chichestera, Martha A. Hassa, John A. Goslingb, Ralph Buttyanc a
Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208-3492, USA Stanford University, Stanford, CA, USA c Columbia University, New York, NY, USA b
Abstract Obstruction can cause changes in bladder structure and function, which may at a certain point in time become irreversible. Both the rabbit and the rat have proven to be excellent models to study the morphological, biochemical and molecular changes that occur in the bladder following obstruction. Similarities between partial out¯ow obstruction in animals and obstructive dysfunction in man include increased bladder mass, increased ®brosis, reduced compliance, increased incidence of detrusor instability and decreased contractile ability. Obstructed bladder function can remain relatively normal for prolonged periods of time, even though bladder mass is increased (compensated stage). If the obstruction is not relieved, bladder function destabilizes and then decompensates, with subsequent risk of serious complications. It is hypothesized that the shift from the compensated to the decompensated stage is related to cyclical periods of ischaemia followed by reperfusion (I/R). This initiates degenerative membrane effects, which supports the process of bladder decompensation. It seems that relief of obstruction during the compensated stage, at least in animals, can induce a rapid and full restoration of the bladder function. However, if the obstruction is relieved during the decompensated phase, bladder function only partially recovers. Irreversible bladder decompensation may be prevented by reducing increased bladder mass and/or by reducing ischaemia/increasing blood supply to the bladder. The antioxidant Vitamin E seems to reduce the progression of decompensation in rabbits. Treatment of rats with the a1-adrenoceptor antagonist doxazosin prior to partial outlet obstruction increases blood ¯ow to the bladder, signi®cantly decreases the effect of obstruction on bladder weight, and signi®cantly protects the contractile function of the obstructed bladder. Pre-treatment of rabbits with the a1adrenoceptor antagonist tamsulosin partly prevents the development of bladder wall hypertrophy due to obstruction. In conclusion, there is evidence that I/R plays an important role in the pathogenesis of obstructive bladder dysfunction. Therefore, I/R should be prevented or relieved. As I/R is the consequence of increased bladder mass following obstruction, therapies that prevent or reduce increased bladder mass (such as the a1-adrenoceptor antagonist tamsulosin) are likely to protect the bladder from (further) dysfunction. Therapies based on both the relief of I/R (increasing blood ¯ow to the bladder, e.g. a1-adrenoceptor antagonists) and preventing I/R induced cellular damage (e.g. antioxidant activity) have been shown experimentally to signi®cantly reduce the severity of bladder dysfunction secondary to partial out¯ow obstruction. Of course, these potentially useful and novel mechanisms of action of therapy should also be investigated in humans. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Obstruction; Bladder; Animal; Bladder wall hypertrophy; a1-adrenoceptor antagonist 1. Introduction Obstructed bladder dysfunction secondary to benign prostatic hyperplasia (BPH), a slowly progressive * Corresponding author. Tel. 1-518-445-7306; Fax: 1-518-445-7248. E-mail address: [email protected] (R.M. Levin).
af¯iction associated with human aging, is characterized by bladder wall hypertrophy (BWH), decreases in urinary ¯ow and compliance, and post-void residual urine volume [1±4]. If the obstruction is not relieved, the bladder decompensates and serious complications such as acute urinary retention, bladder stones, urinary tract infections and renal dysfunction may ultimately
1569-9056/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 5 6 ( 0 2 ) 0 0 1 1 9 - 7
R.M. Levin et al. / European Urology Supplements 1 (2002) 14±20
develop [5]. Treatment often is not sought until the disease is somewhat severe, since bladder function remains relatively ``normal'' as the hypertrophying bladder compensates for the progressive increase in urethral resistance. Recent advances in detection methods (video urodynamics) enable diagnosis of patients with signi®cant lower urinary tract symptoms (LUTS) suggestive of BPH during compensated function, before the bladder becomes dysfunctional (decompensated). The compensated patient can be treated conservatively, surgery is indicated for the decompensated patient (with serious complications) progressing towards irreversible bladder dysfunction. In humans, it is dif®cult to investigate the cellular mechanisms by which obstructive bladder dysfunction occurs. Fortunately, many of the structural and functional changes associated with human bladder pathology can be induced in animal models of partial outlet obstruction [6]. This can be done by placing a ligature or cuff around the urethra. Both the rabbit and the rat have proven to be excellent models to study the pathogenesis of bladder dysfunction secondary to obstruction [7,8]. The goals of our current studies are to identify the cellular and genetic factors that control the bladder's initial response to partial obstruction, regulate the shift from compensated to decompensated function and mediate progressive decompensation. 2. Partial outlet obstruction as a model for obstructed bladder pathology Obstructed bladder dysfunction is characterized by alterations in bladder mass, tissue composition, capacity, compliance and response to pharmacological agents [1±3]. Experimentally, many pathological characteristics of obstructed bladder disease can be reproduced in animal models of partial outlet obstruction [6,9]. The urinary bladder's progressive response to partial outlet obstruction in rats and rabbits can be separated into three stages [6,10±12]. Surgically created mild partial outlet obstruction immediately increases urethral resistance to urine ¯ow resulting in bladder distension (initial stage). Bladder mass increases rapidly during the next 2 weeks, then growth abruptly stops [10,11]. During this stage, areas of focal hypoxia appear within the bladder wall [13,14]. Angiogenesis is stimulated and vascularization and blood ¯ow (BF) increase in proportion to the increasing bladder mass [15±18]. At the end of the initial period, bladder function (ability to generate pressure and to empty) is nearly normal and the bladder enters a compensated stage of
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inde®nite duration, characterized by stabilization of mass and function. At some point, bladder function destabilizes and the organ enters a decompensated stage characterized by progressive deleterious changes, i.e. increasing bladder mass, decreasing contractile responses to electrical ®eld and pharmacological stimulation, decreasing compliance, increasing post-void residual urine volume and increased synthesis and deposition of connective tissue (CT). End-stage decompensation is characterized by CT replacement of smooth muscle (SM) that, ®nally, results in a ®brous organ with little or no contractile function [10,11]. Bladder structure and function recover rapidly and nearly completely if the obstruction is removed during early decompensation [19±21], but only partially recover if the obstruction is released later, i.e. after CT begins to replace SM (note that recovery depends upon severity of decompensation, not duration of obstruction) [22]. 3. Hypothesis on the aetiology of obstructed bladder pathology In response to partial outlet obstruction, acute distension followed by hypoxia induces activation of interconnecting molecular genetic cascades that initially allow the bladder to adapt to the increased urethral resistance, but ultimately lead to degenerative bladder function. Distension- and hypoxia-activated genes [9,13±15] (1) initiate early detrusor SM cell hypertrophy, later induce a switch in SM cell phenotype from a contractile cell to one that synthesizes and secretes collagen, (2) initiate the extracellular matrix (ECM) remodeling that underlies both angiogenesis and ®brosis and (3) hypoxic foci that ®rst appear in the bladder wall during the initial response to obstruction and are present during compensation are the initial sites of the aforementioned genetic changes and of ischaemia/ reperfusion (I/R) induced injury, i.e. Ca2 and reactive oxygen species (ROS) initiated cellular and sub-cellular membrane damage. The shift from compensation to decompensation occurs as these alterations spread into tissue with normal oxygen tension (normoxic tissue). 4. Bladder response to partial outlet obstruction 4.1. Increase in mass In the rat and rabbit, the rapid increase in bladder mass stabilizes after 2 weeks of obstruction [6,10,11] (Fig. 1) at which time bladder function is relatively
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R.M. Levin et al. / European Urology Supplements 1 (2002) 14±20
Fig. 1. Effect of short-term partial outlet obstruction on bladder weight. Each bar is the mean S:E:M: of 6±8 individual rabbits. Please note that bladder weight increases rapidly within the ®rst 2 weeks of obstruction.
normal. The initial increase in bladder mass is due to urothelial hyperplasia, ®broblast hyperplasia, and SM hypertrophy and hyperplasia [10,11]. Results of molecular studies relate these changes to increased expression of basic ®broblast growth factor (bFGF) and a simultaneous decrease in transforming growth factor (TGFb1) transcripts, increased expression of oncogenes including c-myc, Ha-ras and N-ras, and increased transcription of the collagen synthesis genes Cyr-61 and connective tissue growth factor (CTGF) [9,18,23,24]. 4.2. Blood flow and vessel size redistribution (angiogenesis) Relative blood ¯ow (BF) (ml/g) to the rabbit bladder is increased at 1 day post-obstruction, due to vasodilation induced by increased nitric oxide (NO) synthesis and release, then returns to control levels, i.e. BF increases with the increase in bladder mass [17]. Using antibodies to the vascular endothelium marker CD31, we showed that partial outlet obstruction induces a rapid increase and redistribution to small or mid size blood vessels in the bladder wall. Most notable is the marked neovascularization of the obstruction-induced proliferated (``thickened'') serosal compartment (CT) [16,25]. Fig. 2 shows the distribution of vessels according to vessel circumference in control and obstructed animals. Please note that the obstructed bladder exhibits the signi®cantly increased proportion of micro- and small circumference vessels that would be expected after rapid angiogenesis. Results of recent studies (rats) showed that this period of rapid angiogenesis is accompanied by increased expression of hypoxic inducible factor 1-alpha (HIF-1a), angiopoietin and vascular endothelial growth factor (VEGF) [18].
Fig. 2. Effect of partial outlet obstruction on blood vessel circumference. Distribution of blood vessel circumferences in a decompensated bladder compared to a control bladder. Please note that the proportion of microand small-circumference vessels is increased in the decompensated bladder. Each point represents the percentage of vessels which have a speci®c circumference range, i.e. 0.02: >0.01 and <0.03; 0.04: >0.03 and <0.05, etc.
The end of the initial stage is characterized by a rapid reduction in the rate of bladder growth and the cessation of angiogenesis. These events are a direct result of active processes, i.e. the increased expression of the growth suppressor genes P53 and WAF-1 and the appearance of the potent anti-angiogenic factor, angiostatin [9,18]. 4.3. Hypoxic foci Although the bladder's contractile function is relatively normal at the end of the initial stage, focal areas of hypoxia appear during this period [13,14,18]. Tissue hypoxia is revealed using Hypoxyprobe-1 (pimonidazole), a small molecular weight compound that freely diffuses through tissues and forms protein adducts in hypoxic cells only. The Hypoxyprobe-1 protein adducts can be detected in tissue sections immuno - histochemically. Immuno - histochemical staining of a series of transverse sections of rat bladder after partial outlet obstruction showed that hypoxic foci ®rst appear at 3 days post-obstruction in the mucosa and sub-mucosa. By 7 days, these sites shifted to the interstitial spaces and by 14 days signi®cant hypoxic foci were observed within the SM bundles [14]. 4.4. I/R injury Even though relative BF is not reduced at this time, the location of the blood vessels between (and not
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within) the hypertrophied muscle bundles effectively reduces perfusion and oxygen delivery to the enlarged SM cells, the sites of persistent hypoxic foci at 14 days post-obstruction [13]. Results of our studies are consistent with those of Greenland and Brading showing that, in control mini-pigs, BF and oxygenation decreased during bladder contraction (micturition) and that, while BF returned to basal level shortly after the end of the micturition contraction, tissue oxygenation returned more slowly. Partial outlet obstruction, however, resulted in a substantially greater micturition contraction accompanied by a marked reduction in BF during contraction and a signi®cantly prolonged period of severe hypoxia immediately after contraction [26,27]. Temporally, BF recovered while the tissue was still hypoxic; both BF and oxygenation recovered more slowly after micturition contraction than they did in the control bladders. Functionally, cyclic I/R within the SM compartment results in the release of intracellular free calcium ([Ca2]i), leading to Ca2 overload, and the generation and release of ROS. These processes, in turn, cause the activation of Ca2-activated phospholipases, such as phospholipase A2 (PLA2) [28], and proteases, such as calpain [29] as well as causing reactive oxygen species (ROS) injury such as protein (thiol) oxidation and membrane lipid peroxidation [30]. The consequence of these activities is progressive damage to nerve, synaptic and intracellular membranes, including those of mitochondria and the sarcoplasmatic reticulum (SR) [8,29]. These degenerative membrane effects initiate and support the process of bladder decompensation, which in its end-stage results in irreversible metabolic and contractile dysfunctions. The end result of decompensation is either a bladder with a thick ®brous wall, low capacity, poor compliance and little or no contractile function, or a dilated bladder with a thin ®brous wall, high capacity and little or no contractile function (Fig. 3). Results of electron microscopy studies of obstructed rabbit bladders and of bladder tissue from men with signi®cant obstructive symptoms show that the level of membrane damage observed was related directly to the level of contractile dysfunction measured and that membrane damage was, characteristically, consistent with an I/R aetiology [8,31]. As bladder growth continues, decompensation progresses. Angiogenesis, however, no longer occurs apace with bladder growth due to the presence of angiostatin and, possibly, other angiostatic factors [18] resulting in a progressive decrease in relative BF that is proportional to the level of decompensation present (Fig. 4) [32].
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Fig. 3. Hypothesis on the aetiology of bladder decompensation.
5. Potential therapies to prevent bladder decompensation If bladder decompensation is directly related to the magnitude of the increase in bladder mass (wall thickness), decreased detrusor BF/ischaemia and the related increase in the activities of hydrolytic and degradative enzymes, then therapies directed at reducing the rate of bladder growth, improving BF and bladder perfusion,
Fig. 4. Effect of chronic partial outlet obstruction on blood ¯ow to the mucosa and muscle of the rabbit bladder. Blood ¯ow was quantitated using a ¯uorescent microsphere methodology. Each bar represents the mean of 4±6 individual rabbits. Please note that the decreased blood ¯ow to the smooth muscle is proportional to the level of decompensation. signi®cantly different from control, p < 0:05.
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Fig. 5. Effect of doxazosin on the response of male rats to partial outlet obstruction. Each bar is the mean S.E.M. of 4±8 individual rats. Blood ¯ow was quantitated using a ¯uorescent microsphere methodology. signi®cantly different from control, p < 0:05; # signi®cantly different from vehicle treatment, p < 0:05.
and protecting cellular and sub-cellular membranes from activated hydrolytic enzymes should effectively reduce the rate of decompensation and improve bladder function in the presence of increased urethral resistance. 5.1. Antioxidants Feeding rabbits, a diet supplemented with the lipophilic antioxidant Vitamin E, for 4 weeks before creating a partial outlet obstruction, signi®cantly preserved bladder contractility [33]. Thus, protecting cellular and sub-cellular membranes from ROS-induced damage proved effective in reducing the progression of bladder decompensation. Similarly, natural products with high antioxidant activity also protected the bladder from obstructive damage [34,35]. 5.2. a1 -adrenoceptor antagonists a1-adrenoceptor (AR) antagonists such as tamsulosin and doxazosin are well known therapeutics for obstructed LUTS patients. Although their mechanism of action is thought to be, primarily, inhibition of prostatic a1-AR-stimulated SM contraction there may be additional mechanisms responsible for the effectiveness of these drugs. Since BF to a tissue is regulated, in part, by a1-AR tone of arterioles, a1-AR antagonists may act, in part, by regulating bladder BF. In a recent experiment, doxazosin (30 mg/kg) was administered orally to rats for 4 weeks before creating partial outlet obstruction. After 2 weeks of obstruction the following parameters were measured: bladder weight, relative BF using ¯uorescent microspheres, and contractile responses of isolated bladder strips to
®eld stimulation (FS), carbachol, ATP, and KCl [36]. Four weeks pre-treatment with doxazosin signi®cantly increased bladder BF in control and obstructed rats (Fig. 5). The magnitude of the increased bladder weight in the vehicle-treated obstructed group was signi®cantly greater than in the doxazosin-treated obstructed group (Fig. 5). Partial outlet obstruction resulted in signi®cant decreases in the contractile responses to FS of bladders from treated and nontreated rats, however, the magnitude of the decreased response of the bladders from non-treated rats was signi®cantly greater. The response to KCl was signi®cantly reduced by obstruction in the vehicle-treated group but not in the doxazosin-treated group. It is interesting to note that preliminary ®ndings in LUTS/BPH patients demonstrated that tamsulosin increases blood ¯ow to both prostate and bladder and increases bladder capacity [37].
Fig. 6. Effect of tamsulosin on the response of male rabbits to partial outlet obstruction. signi®cantly different from control, p < 0:05.
R.M. Levin et al. / European Urology Supplements 1 (2002) 14±20
In a similar study, rabbits were administered tamsulosin before creating partial outlet obstruction. Two groups of ®ve obstructed animals were pretreated with either tamsulosin (28 mg/kg h) or a vehicle for 1 week. Five sham operated animals served as control. The results show that the bladder weights of obstructed rabbits treated with vehicle (6:3 4:0 g) were higher than in obstructed rabbits treated with tamsulosin (3:92 1:4 g) compared to 2:35 0:5 g in the controls (Fig. 6) [38]. This shows that pretreatment of tamsulosin partly prevented increased bladder mass in these animals which may preserve bladder blood ¯ow and reduce I/R and its subsequent bladder damage.
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6. Conclusions There is increasing evidence that obstructed bladder disease is related directly to decreased perfusion/ ischaemia subsequent to increased bladder mass resulting in detrusor hypoxia and ROS injury [8]. Results of current studies indicate that agents that are targeted at preventing increased bladder mass, improving detrusor perfusion and reducing I/R injury effectively reduce obstruction-induced contractile dysfunction. The mechanisms of action of a1-AR antagonists such as doxazosin and tamsulosin may include the improvement of bladder perfusion as well as the reduction of intra-prostatic tension.
References [1] Grayhack JT, Kozlowski JM. Benign prostatic hyperplasia. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JD, editors. Adult and pediatric urology, 3rd ed. Chicago: Mosby Year Book Medical Publishers; 1996. p. 1501±74. [2] Barry MJ, Meigs JB. The natural history of benign prostatic hyperplasia. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 106±15. [3] Girman CJ, Guess HA. Epidemiology of benign prostatic hyperplasia. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 106±15. [4] Zderic SA, Levin RM, Wein AJ. Voiding function and dysfunction: a relevant anatomy, physiology, and pharmacology, and molecular biology. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JD, editors. Adult and pediatric urology, 3rd ed. Chicago: Mosby Year Book Medical Publishers; 1996. p. 1159±219. [5] Anderson JB, Roehrborn CG, Schalken JA, Emberton M. The progression of benign prostatic hyperplasia: examining the evidence and determining the risk. Eur Urol 2001;39:390±9. [6] Levin RM, Brading AF, Mills IW, Longhust PA. Experimental models of bladder obstruction. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 169±96. [7] Levin RM, Levin SS, Zhao Y, Buttyan R. Cellular and molecular aspects of bladder hypertrophy. Eur Urol 1997;32(Suppl 1): 15±21. [8] Levin RM, Haugaard N, O'Connor L, Buttyan R, Das AK, Dixon JS, Gosling JA. Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 2000;19:609±29. [9] Buttyan R, Chen MW, Levin RM. Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction. Eur Urol 1997;32(Suppl):32±9. [10] Levin RM, Haugaard N, Levin SS, Buttyan R, Chen M-W, Monson FC, Wein AJ. Bladder function in experimental outlet obstruction: pharmacologic responses to alterations in innervation, energetics, calcium mobilization, and genetics. In: Zderic S, editor. Muscle, matrix, and bladder function. New York: Plenum Press; 1995. p. 7±19. [11] Levin RM, Longhurst PA, Monson FC, Haugaard N, Wein AJ. Experimental studies on bladder outlet obstruction. In: Lepor H, Lawson RK, editors. Prostate diseases. Philadelphia: Saunders; 1993. p. 119±30. [12] Malkowicz SB, Wein AJ, Elbadawi A, Van Arsdalen K, Ruggieri MR, Levin RM. Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. J Urol 1986;136: 1324±9.
[13] Ghafar MA, Shabsigh A, Chichester P, Anastasiadis AG, Levin RM, Buttyan R. Effects of chronic partial outlet obstruction on blood ¯ow and oxygenation of the rat bladder. J Urol 2002;167: 1508±12. [14] Levin RM, O'Connor LJ, Leggett RE, Whitbeck C, Chichester P. Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation. Neurourol Urodyn [in press]. [15] Shabsigh A, Hayek OH, Weiner D, Saidi J, Kaplan SA, Kiss A, et al. Acute increase in blood ¯ow to the rat bladder subsequent to partial bladder outlet obstruction. Neurourol Urodyn 2000;19: 195±208. [16] Chichester P, Lieb J, Levin SS, Buttyan R, Horan P, Levin RM. Vascular response of the rabbit bladder to short-term partial outlet obstruction. Mol Cell Biochem 2000;208:19±26. [17] Lieb J, Chichester P, Kogan B, Das AK, Leggett RE, Schroeder A, Levin RM. Rabbit urinary bladder blood ¯ow changes during the initial stage of partial outlet obstruction. J Urol 2000;164: 1390±7. [18] Ghafar MA, Anastasiadis AG, Olsson LE, Chichester P, Kaplan SA, Buttyan R, Levin RM. Hypoxia and an angiogenic response in the partially obstructed rat bladder. Lab Invest 2002;82:903±9. [19] Levin RM, Malkowicz SB, Wein AJ, Atta MA, Elbadawi A. Recovery from short term obstruction of the rabbit urinary bladder. J Urol 1985;134:388±90. [20] Wang Z, Levin RM, Chacko S. Expression of smooth muscle myosin isoforms in urinary bladder smooth muscle during hypertrophy and regression. Lab Invest 1995;73:244±51. [21] Gabella G, Uvelius B. Reversal of muscle hypertrophy in the rat urinary bladder after removal of urethral obstruction. Cell Tissue Res 1994;277(2):333±9. [22] Nevel-McGarvey CA, Levin RM, Haugaard N, Wu X, Hudson AP. Mitochondrial and mitochondria-related nuclear genetic function in rabbit urinary bladder following reversal of outlet obstruction. Mol Cell Biochem 1999;197:161±72. [23] Buttyan R, Jacobs B, Blaivis JG, Levin RM. The early molecular response to rabbit bladder outlet obstruction. Neurourol Urodyn 1992;11:253±60. [24] Chaqour B, Whitbeck C, Horan P, Levin RM. Cysteine-rich 61 (Cyr61) and Connective Tissue Growth Factor (CTGF) gene expression in detrusor bladder is an early marker of phenotypical remodeling after outlet obstruction in the rat model [submitted for publication]. [25] Chichester P, Schroder A, Horan P, Levin RM. Vascular response of the rabbit bladder to chronic partial outlet obstruction. Mol Cell Biochem 2001;226:1±8.
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[26] Greenland JE, Hvistendahl JJ, Andersen H, Jorgensen TM, McMurray G, Cortina-Borja M, et al. The effect of bladder outlet obstruction on tissue oxygen tension and blood ¯ow in the pig bladder. BJU Int 2000;85:1109±14. [27] Greenland JE, Brading AF. The effect of bladder out¯ow obstruction on detrusor blood ¯ow changes during the voiding cycle in conscious pigs. J Urol 2001;165:245±8. [28] Masick JM, Levin RM, Hass MA. Prostaglandin levels in the smooth muscle and mucosa of the partially obstructed rabbit urinary bladder. Prostaglandins Other Lipid Mediat 2001;66:211±9. [29] Zhao Y, Levin SS, Wein AJ, Levin RM. Correlation of ischemia/ reperfusion and partial outlet obstruction induced spectrin proteolysis by calpain with contractile dysfunction in the rabbit bladder. Urology 1997;49:293±300. [30] Bauer V, Sotnikova R, Machova J, Matyas S, Pucovsky V, Stefek M. Reactive oxygen species induced smooth muscle responses in the intestine, vessels and airways and the effect of antioxidants. Life Sci 1999;65:1909±17. [31] Gosling JA, Kung LS, Dixon JS, Horan P, Whitbeck C, Levin RM. Correlation between the structure and function of the urinary bladder following partial outlet obstruction. J Urol 2000;163: 1349±56.
[32] SchroÈder A, Chichester P, Kogan BA, Longhurst PA, Lieb J, Levin, RM. Effect of chronic bladder outlet obstruction on the blood ¯ow of the rabbit urinary bladder. J Urol 165:640±6. [33] Parekh MH, Lobel R, O'Connor LJ, Leggett RE, Levin RM. Protective effect of vitamin E on the response of the rabbit bladder to partial outlet obstruction. J Urol 2001;166:341±6. [34] Levin RM, Das AK, Haugaard N, Novitsky Y, Horan P, Leggett RE, et al. Bene®cial effects of Tadenan therapy following two weeks of partial outlet obstruction in the rabbit. Neurourol Urodyn 1997;16: 583±99. [35] Levin RM, Kawashima Y, Mizutani K, Leggett RE, Whitbeck C, Horan P. Effect of oral Kohki tea on the bladder dysfunctions induced by severe partial outlet obstruction. J Urol 2002;167:2260-6. [36] Das AK, Leggett RE, Whitbeck C, Horan P, Levin RM. Effect of doxazosin on bladder function following partial outlet obstruction in rats. Neurourol Urodyn 2002;21:160±6. [37] Pinggera GM, Schuster A, Pallwein L, Klauser A, Frauscher F, FurtmuÈller B. Alpha-blockers increase vesical and prostatic blood ¯ow and bladder capacity. J Urol 2002;167(4 Suppl):373 [Abstract 1485]. [38] Korstanje C, de Wijn S, Huisman A, Koorn J, van den Hoven W. Tamsulosin and bladder hypertrophy in a rabbit model. Eur Urol Suppl 2002;1(1):61 [Abstract 233].
European Urology Supplements 1 (2002) 14±20
Obstructive Bladder Dysfunction: Morphological, Biochemical and Molecular Changes Robert M. Levina,*, Paul Chichestera, Martha A. Hassa, John A. Goslingb, Ralph Buttyanc a
Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208-3492, USA Stanford University, Stanford, CA, USA c Columbia University, New York, NY, USA b
Abstract Obstruction can cause changes in bladder structure and function, which may at a certain point in time become irreversible. Both the rabbit and the rat have proven to be excellent models to study the morphological, biochemical and molecular changes that occur in the bladder following obstruction. Similarities between partial out¯ow obstruction in animals and obstructive dysfunction in man include increased bladder mass, increased ®brosis, reduced compliance, increased incidence of detrusor instability and decreased contractile ability. Obstructed bladder function can remain relatively normal for prolonged periods of time, even though bladder mass is increased (compensated stage). If the obstruction is not relieved, bladder function destabilizes and then decompensates, with subsequent risk of serious complications. It is hypothesized that the shift from the compensated to the decompensated stage is related to cyclical periods of ischaemia followed by reperfusion (I/R). This initiates degenerative membrane effects, which supports the process of bladder decompensation. It seems that relief of obstruction during the compensated stage, at least in animals, can induce a rapid and full restoration of the bladder function. However, if the obstruction is relieved during the decompensated phase, bladder function only partially recovers. Irreversible bladder decompensation may be prevented by reducing increased bladder mass and/or by reducing ischaemia/increasing blood supply to the bladder. The antioxidant Vitamin E seems to reduce the progression of decompensation in rabbits. Treatment of rats with the a1-adrenoceptor antagonist doxazosin prior to partial outlet obstruction increases blood ¯ow to the bladder, signi®cantly decreases the effect of obstruction on bladder weight, and signi®cantly protects the contractile function of the obstructed bladder. Pre-treatment of rabbits with the a1adrenoceptor antagonist tamsulosin partly prevents the development of bladder wall hypertrophy due to obstruction. In conclusion, there is evidence that I/R plays an important role in the pathogenesis of obstructive bladder dysfunction. Therefore, I/R should be prevented or relieved. As I/R is the consequence of increased bladder mass following obstruction, therapies that prevent or reduce increased bladder mass (such as the a1-adrenoceptor antagonist tamsulosin) are likely to protect the bladder from (further) dysfunction. Therapies based on both the relief of I/R (increasing blood ¯ow to the bladder, e.g. a1-adrenoceptor antagonists) and preventing I/R induced cellular damage (e.g. antioxidant activity) have been shown experimentally to signi®cantly reduce the severity of bladder dysfunction secondary to partial out¯ow obstruction. Of course, these potentially useful and novel mechanisms of action of therapy should also be investigated in humans. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Obstruction; Bladder; Animal; Bladder wall hypertrophy; a1-adrenoceptor antagonist 1. Introduction Obstructed bladder dysfunction secondary to benign prostatic hyperplasia (BPH), a slowly progressive * Corresponding author. Tel. 1-518-445-7306; Fax: 1-518-445-7248. E-mail address: [email protected] (R.M. Levin).
af¯iction associated with human aging, is characterized by bladder wall hypertrophy (BWH), decreases in urinary ¯ow and compliance, and post-void residual urine volume [1±4]. If the obstruction is not relieved, the bladder decompensates and serious complications such as acute urinary retention, bladder stones, urinary tract infections and renal dysfunction may ultimately
1569-9056/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 5 6 ( 0 2 ) 0 0 1 1 9 - 7
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develop [5]. Treatment often is not sought until the disease is somewhat severe, since bladder function remains relatively ``normal'' as the hypertrophying bladder compensates for the progressive increase in urethral resistance. Recent advances in detection methods (video urodynamics) enable diagnosis of patients with signi®cant lower urinary tract symptoms (LUTS) suggestive of BPH during compensated function, before the bladder becomes dysfunctional (decompensated). The compensated patient can be treated conservatively, surgery is indicated for the decompensated patient (with serious complications) progressing towards irreversible bladder dysfunction. In humans, it is dif®cult to investigate the cellular mechanisms by which obstructive bladder dysfunction occurs. Fortunately, many of the structural and functional changes associated with human bladder pathology can be induced in animal models of partial outlet obstruction [6]. This can be done by placing a ligature or cuff around the urethra. Both the rabbit and the rat have proven to be excellent models to study the pathogenesis of bladder dysfunction secondary to obstruction [7,8]. The goals of our current studies are to identify the cellular and genetic factors that control the bladder's initial response to partial obstruction, regulate the shift from compensated to decompensated function and mediate progressive decompensation. 2. Partial outlet obstruction as a model for obstructed bladder pathology Obstructed bladder dysfunction is characterized by alterations in bladder mass, tissue composition, capacity, compliance and response to pharmacological agents [1±3]. Experimentally, many pathological characteristics of obstructed bladder disease can be reproduced in animal models of partial outlet obstruction [6,9]. The urinary bladder's progressive response to partial outlet obstruction in rats and rabbits can be separated into three stages [6,10±12]. Surgically created mild partial outlet obstruction immediately increases urethral resistance to urine ¯ow resulting in bladder distension (initial stage). Bladder mass increases rapidly during the next 2 weeks, then growth abruptly stops [10,11]. During this stage, areas of focal hypoxia appear within the bladder wall [13,14]. Angiogenesis is stimulated and vascularization and blood ¯ow (BF) increase in proportion to the increasing bladder mass [15±18]. At the end of the initial period, bladder function (ability to generate pressure and to empty) is nearly normal and the bladder enters a compensated stage of
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inde®nite duration, characterized by stabilization of mass and function. At some point, bladder function destabilizes and the organ enters a decompensated stage characterized by progressive deleterious changes, i.e. increasing bladder mass, decreasing contractile responses to electrical ®eld and pharmacological stimulation, decreasing compliance, increasing post-void residual urine volume and increased synthesis and deposition of connective tissue (CT). End-stage decompensation is characterized by CT replacement of smooth muscle (SM) that, ®nally, results in a ®brous organ with little or no contractile function [10,11]. Bladder structure and function recover rapidly and nearly completely if the obstruction is removed during early decompensation [19±21], but only partially recover if the obstruction is released later, i.e. after CT begins to replace SM (note that recovery depends upon severity of decompensation, not duration of obstruction) [22]. 3. Hypothesis on the aetiology of obstructed bladder pathology In response to partial outlet obstruction, acute distension followed by hypoxia induces activation of interconnecting molecular genetic cascades that initially allow the bladder to adapt to the increased urethral resistance, but ultimately lead to degenerative bladder function. Distension- and hypoxia-activated genes [9,13±15] (1) initiate early detrusor SM cell hypertrophy, later induce a switch in SM cell phenotype from a contractile cell to one that synthesizes and secretes collagen, (2) initiate the extracellular matrix (ECM) remodeling that underlies both angiogenesis and ®brosis and (3) hypoxic foci that ®rst appear in the bladder wall during the initial response to obstruction and are present during compensation are the initial sites of the aforementioned genetic changes and of ischaemia/ reperfusion (I/R) induced injury, i.e. Ca2 and reactive oxygen species (ROS) initiated cellular and sub-cellular membrane damage. The shift from compensation to decompensation occurs as these alterations spread into tissue with normal oxygen tension (normoxic tissue). 4. Bladder response to partial outlet obstruction 4.1. Increase in mass In the rat and rabbit, the rapid increase in bladder mass stabilizes after 2 weeks of obstruction [6,10,11] (Fig. 1) at which time bladder function is relatively
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Fig. 1. Effect of short-term partial outlet obstruction on bladder weight. Each bar is the mean S:E:M: of 6±8 individual rabbits. Please note that bladder weight increases rapidly within the ®rst 2 weeks of obstruction.
normal. The initial increase in bladder mass is due to urothelial hyperplasia, ®broblast hyperplasia, and SM hypertrophy and hyperplasia [10,11]. Results of molecular studies relate these changes to increased expression of basic ®broblast growth factor (bFGF) and a simultaneous decrease in transforming growth factor (TGFb1) transcripts, increased expression of oncogenes including c-myc, Ha-ras and N-ras, and increased transcription of the collagen synthesis genes Cyr-61 and connective tissue growth factor (CTGF) [9,18,23,24]. 4.2. Blood flow and vessel size redistribution (angiogenesis) Relative blood ¯ow (BF) (ml/g) to the rabbit bladder is increased at 1 day post-obstruction, due to vasodilation induced by increased nitric oxide (NO) synthesis and release, then returns to control levels, i.e. BF increases with the increase in bladder mass [17]. Using antibodies to the vascular endothelium marker CD31, we showed that partial outlet obstruction induces a rapid increase and redistribution to small or mid size blood vessels in the bladder wall. Most notable is the marked neovascularization of the obstruction-induced proliferated (``thickened'') serosal compartment (CT) [16,25]. Fig. 2 shows the distribution of vessels according to vessel circumference in control and obstructed animals. Please note that the obstructed bladder exhibits the signi®cantly increased proportion of micro- and small circumference vessels that would be expected after rapid angiogenesis. Results of recent studies (rats) showed that this period of rapid angiogenesis is accompanied by increased expression of hypoxic inducible factor 1-alpha (HIF-1a), angiopoietin and vascular endothelial growth factor (VEGF) [18].
Fig. 2. Effect of partial outlet obstruction on blood vessel circumference. Distribution of blood vessel circumferences in a decompensated bladder compared to a control bladder. Please note that the proportion of microand small-circumference vessels is increased in the decompensated bladder. Each point represents the percentage of vessels which have a speci®c circumference range, i.e. 0.02: >0.01 and <0.03; 0.04: >0.03 and <0.05, etc.
The end of the initial stage is characterized by a rapid reduction in the rate of bladder growth and the cessation of angiogenesis. These events are a direct result of active processes, i.e. the increased expression of the growth suppressor genes P53 and WAF-1 and the appearance of the potent anti-angiogenic factor, angiostatin [9,18]. 4.3. Hypoxic foci Although the bladder's contractile function is relatively normal at the end of the initial stage, focal areas of hypoxia appear during this period [13,14,18]. Tissue hypoxia is revealed using Hypoxyprobe-1 (pimonidazole), a small molecular weight compound that freely diffuses through tissues and forms protein adducts in hypoxic cells only. The Hypoxyprobe-1 protein adducts can be detected in tissue sections immuno - histochemically. Immuno - histochemical staining of a series of transverse sections of rat bladder after partial outlet obstruction showed that hypoxic foci ®rst appear at 3 days post-obstruction in the mucosa and sub-mucosa. By 7 days, these sites shifted to the interstitial spaces and by 14 days signi®cant hypoxic foci were observed within the SM bundles [14]. 4.4. I/R injury Even though relative BF is not reduced at this time, the location of the blood vessels between (and not
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within) the hypertrophied muscle bundles effectively reduces perfusion and oxygen delivery to the enlarged SM cells, the sites of persistent hypoxic foci at 14 days post-obstruction [13]. Results of our studies are consistent with those of Greenland and Brading showing that, in control mini-pigs, BF and oxygenation decreased during bladder contraction (micturition) and that, while BF returned to basal level shortly after the end of the micturition contraction, tissue oxygenation returned more slowly. Partial outlet obstruction, however, resulted in a substantially greater micturition contraction accompanied by a marked reduction in BF during contraction and a signi®cantly prolonged period of severe hypoxia immediately after contraction [26,27]. Temporally, BF recovered while the tissue was still hypoxic; both BF and oxygenation recovered more slowly after micturition contraction than they did in the control bladders. Functionally, cyclic I/R within the SM compartment results in the release of intracellular free calcium ([Ca2]i), leading to Ca2 overload, and the generation and release of ROS. These processes, in turn, cause the activation of Ca2-activated phospholipases, such as phospholipase A2 (PLA2) [28], and proteases, such as calpain [29] as well as causing reactive oxygen species (ROS) injury such as protein (thiol) oxidation and membrane lipid peroxidation [30]. The consequence of these activities is progressive damage to nerve, synaptic and intracellular membranes, including those of mitochondria and the sarcoplasmatic reticulum (SR) [8,29]. These degenerative membrane effects initiate and support the process of bladder decompensation, which in its end-stage results in irreversible metabolic and contractile dysfunctions. The end result of decompensation is either a bladder with a thick ®brous wall, low capacity, poor compliance and little or no contractile function, or a dilated bladder with a thin ®brous wall, high capacity and little or no contractile function (Fig. 3). Results of electron microscopy studies of obstructed rabbit bladders and of bladder tissue from men with signi®cant obstructive symptoms show that the level of membrane damage observed was related directly to the level of contractile dysfunction measured and that membrane damage was, characteristically, consistent with an I/R aetiology [8,31]. As bladder growth continues, decompensation progresses. Angiogenesis, however, no longer occurs apace with bladder growth due to the presence of angiostatin and, possibly, other angiostatic factors [18] resulting in a progressive decrease in relative BF that is proportional to the level of decompensation present (Fig. 4) [32].
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Fig. 3. Hypothesis on the aetiology of bladder decompensation.
5. Potential therapies to prevent bladder decompensation If bladder decompensation is directly related to the magnitude of the increase in bladder mass (wall thickness), decreased detrusor BF/ischaemia and the related increase in the activities of hydrolytic and degradative enzymes, then therapies directed at reducing the rate of bladder growth, improving BF and bladder perfusion,
Fig. 4. Effect of chronic partial outlet obstruction on blood ¯ow to the mucosa and muscle of the rabbit bladder. Blood ¯ow was quantitated using a ¯uorescent microsphere methodology. Each bar represents the mean of 4±6 individual rabbits. Please note that the decreased blood ¯ow to the smooth muscle is proportional to the level of decompensation. signi®cantly different from control, p < 0:05.
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Fig. 5. Effect of doxazosin on the response of male rats to partial outlet obstruction. Each bar is the mean S.E.M. of 4±8 individual rats. Blood ¯ow was quantitated using a ¯uorescent microsphere methodology. signi®cantly different from control, p < 0:05; # signi®cantly different from vehicle treatment, p < 0:05.
and protecting cellular and sub-cellular membranes from activated hydrolytic enzymes should effectively reduce the rate of decompensation and improve bladder function in the presence of increased urethral resistance. 5.1. Antioxidants Feeding rabbits, a diet supplemented with the lipophilic antioxidant Vitamin E, for 4 weeks before creating a partial outlet obstruction, signi®cantly preserved bladder contractility [33]. Thus, protecting cellular and sub-cellular membranes from ROS-induced damage proved effective in reducing the progression of bladder decompensation. Similarly, natural products with high antioxidant activity also protected the bladder from obstructive damage [34,35]. 5.2. a1 -adrenoceptor antagonists a1-adrenoceptor (AR) antagonists such as tamsulosin and doxazosin are well known therapeutics for obstructed LUTS patients. Although their mechanism of action is thought to be, primarily, inhibition of prostatic a1-AR-stimulated SM contraction there may be additional mechanisms responsible for the effectiveness of these drugs. Since BF to a tissue is regulated, in part, by a1-AR tone of arterioles, a1-AR antagonists may act, in part, by regulating bladder BF. In a recent experiment, doxazosin (30 mg/kg) was administered orally to rats for 4 weeks before creating partial outlet obstruction. After 2 weeks of obstruction the following parameters were measured: bladder weight, relative BF using ¯uorescent microspheres, and contractile responses of isolated bladder strips to
®eld stimulation (FS), carbachol, ATP, and KCl [36]. Four weeks pre-treatment with doxazosin signi®cantly increased bladder BF in control and obstructed rats (Fig. 5). The magnitude of the increased bladder weight in the vehicle-treated obstructed group was signi®cantly greater than in the doxazosin-treated obstructed group (Fig. 5). Partial outlet obstruction resulted in signi®cant decreases in the contractile responses to FS of bladders from treated and nontreated rats, however, the magnitude of the decreased response of the bladders from non-treated rats was signi®cantly greater. The response to KCl was signi®cantly reduced by obstruction in the vehicle-treated group but not in the doxazosin-treated group. It is interesting to note that preliminary ®ndings in LUTS/BPH patients demonstrated that tamsulosin increases blood ¯ow to both prostate and bladder and increases bladder capacity [37].
Fig. 6. Effect of tamsulosin on the response of male rabbits to partial outlet obstruction. signi®cantly different from control, p < 0:05.
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In a similar study, rabbits were administered tamsulosin before creating partial outlet obstruction. Two groups of ®ve obstructed animals were pretreated with either tamsulosin (28 mg/kg h) or a vehicle for 1 week. Five sham operated animals served as control. The results show that the bladder weights of obstructed rabbits treated with vehicle (6:3 4:0 g) were higher than in obstructed rabbits treated with tamsulosin (3:92 1:4 g) compared to 2:35 0:5 g in the controls (Fig. 6) [38]. This shows that pretreatment of tamsulosin partly prevented increased bladder mass in these animals which may preserve bladder blood ¯ow and reduce I/R and its subsequent bladder damage.
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6. Conclusions There is increasing evidence that obstructed bladder disease is related directly to decreased perfusion/ ischaemia subsequent to increased bladder mass resulting in detrusor hypoxia and ROS injury [8]. Results of current studies indicate that agents that are targeted at preventing increased bladder mass, improving detrusor perfusion and reducing I/R injury effectively reduce obstruction-induced contractile dysfunction. The mechanisms of action of a1-AR antagonists such as doxazosin and tamsulosin may include the improvement of bladder perfusion as well as the reduction of intra-prostatic tension.
References [1] Grayhack JT, Kozlowski JM. Benign prostatic hyperplasia. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JD, editors. Adult and pediatric urology, 3rd ed. Chicago: Mosby Year Book Medical Publishers; 1996. p. 1501±74. [2] Barry MJ, Meigs JB. The natural history of benign prostatic hyperplasia. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 106±15. [3] Girman CJ, Guess HA. Epidemiology of benign prostatic hyperplasia. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 106±15. [4] Zderic SA, Levin RM, Wein AJ. Voiding function and dysfunction: a relevant anatomy, physiology, and pharmacology, and molecular biology. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JD, editors. Adult and pediatric urology, 3rd ed. Chicago: Mosby Year Book Medical Publishers; 1996. p. 1159±219. [5] Anderson JB, Roehrborn CG, Schalken JA, Emberton M. The progression of benign prostatic hyperplasia: examining the evidence and determining the risk. Eur Urol 2001;39:390±9. [6] Levin RM, Brading AF, Mills IW, Longhust PA. Experimental models of bladder obstruction. In: Lepor H, editor. Prostatic diseases. Philadelphia: Saunders; 2000. p. 169±96. [7] Levin RM, Levin SS, Zhao Y, Buttyan R. Cellular and molecular aspects of bladder hypertrophy. Eur Urol 1997;32(Suppl 1): 15±21. [8] Levin RM, Haugaard N, O'Connor L, Buttyan R, Das AK, Dixon JS, Gosling JA. Obstructive response of human bladder to BPH vs. rabbit bladder response to partial outlet obstruction: a direct comparison. Neurourol Urodyn 2000;19:609±29. [9] Buttyan R, Chen MW, Levin RM. Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction. Eur Urol 1997;32(Suppl):32±9. [10] Levin RM, Haugaard N, Levin SS, Buttyan R, Chen M-W, Monson FC, Wein AJ. Bladder function in experimental outlet obstruction: pharmacologic responses to alterations in innervation, energetics, calcium mobilization, and genetics. In: Zderic S, editor. Muscle, matrix, and bladder function. New York: Plenum Press; 1995. p. 7±19. [11] Levin RM, Longhurst PA, Monson FC, Haugaard N, Wein AJ. Experimental studies on bladder outlet obstruction. In: Lepor H, Lawson RK, editors. Prostate diseases. Philadelphia: Saunders; 1993. p. 119±30. [12] Malkowicz SB, Wein AJ, Elbadawi A, Van Arsdalen K, Ruggieri MR, Levin RM. Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. J Urol 1986;136: 1324±9.
[13] Ghafar MA, Shabsigh A, Chichester P, Anastasiadis AG, Levin RM, Buttyan R. Effects of chronic partial outlet obstruction on blood ¯ow and oxygenation of the rat bladder. J Urol 2002;167: 1508±12. [14] Levin RM, O'Connor LJ, Leggett RE, Whitbeck C, Chichester P. Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation. Neurourol Urodyn [in press]. [15] Shabsigh A, Hayek OH, Weiner D, Saidi J, Kaplan SA, Kiss A, et al. Acute increase in blood ¯ow to the rat bladder subsequent to partial bladder outlet obstruction. Neurourol Urodyn 2000;19: 195±208. [16] Chichester P, Lieb J, Levin SS, Buttyan R, Horan P, Levin RM. Vascular response of the rabbit bladder to short-term partial outlet obstruction. Mol Cell Biochem 2000;208:19±26. [17] Lieb J, Chichester P, Kogan B, Das AK, Leggett RE, Schroeder A, Levin RM. Rabbit urinary bladder blood ¯ow changes during the initial stage of partial outlet obstruction. J Urol 2000;164: 1390±7. [18] Ghafar MA, Anastasiadis AG, Olsson LE, Chichester P, Kaplan SA, Buttyan R, Levin RM. Hypoxia and an angiogenic response in the partially obstructed rat bladder. Lab Invest 2002;82:903±9. [19] Levin RM, Malkowicz SB, Wein AJ, Atta MA, Elbadawi A. Recovery from short term obstruction of the rabbit urinary bladder. J Urol 1985;134:388±90. [20] Wang Z, Levin RM, Chacko S. Expression of smooth muscle myosin isoforms in urinary bladder smooth muscle during hypertrophy and regression. Lab Invest 1995;73:244±51. [21] Gabella G, Uvelius B. Reversal of muscle hypertrophy in the rat urinary bladder after removal of urethral obstruction. Cell Tissue Res 1994;277(2):333±9. [22] Nevel-McGarvey CA, Levin RM, Haugaard N, Wu X, Hudson AP. Mitochondrial and mitochondria-related nuclear genetic function in rabbit urinary bladder following reversal of outlet obstruction. Mol Cell Biochem 1999;197:161±72. [23] Buttyan R, Jacobs B, Blaivis JG, Levin RM. The early molecular response to rabbit bladder outlet obstruction. Neurourol Urodyn 1992;11:253±60. [24] Chaqour B, Whitbeck C, Horan P, Levin RM. Cysteine-rich 61 (Cyr61) and Connective Tissue Growth Factor (CTGF) gene expression in detrusor bladder is an early marker of phenotypical remodeling after outlet obstruction in the rat model [submitted for publication]. [25] Chichester P, Schroder A, Horan P, Levin RM. Vascular response of the rabbit bladder to chronic partial outlet obstruction. Mol Cell Biochem 2001;226:1±8.
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[26] Greenland JE, Hvistendahl JJ, Andersen H, Jorgensen TM, McMurray G, Cortina-Borja M, et al. The effect of bladder outlet obstruction on tissue oxygen tension and blood ¯ow in the pig bladder. BJU Int 2000;85:1109±14. [27] Greenland JE, Brading AF. The effect of bladder out¯ow obstruction on detrusor blood ¯ow changes during the voiding cycle in conscious pigs. J Urol 2001;165:245±8. [28] Masick JM, Levin RM, Hass MA. Prostaglandin levels in the smooth muscle and mucosa of the partially obstructed rabbit urinary bladder. Prostaglandins Other Lipid Mediat 2001;66:211±9. [29] Zhao Y, Levin SS, Wein AJ, Levin RM. Correlation of ischemia/ reperfusion and partial outlet obstruction induced spectrin proteolysis by calpain with contractile dysfunction in the rabbit bladder. Urology 1997;49:293±300. [30] Bauer V, Sotnikova R, Machova J, Matyas S, Pucovsky V, Stefek M. Reactive oxygen species induced smooth muscle responses in the intestine, vessels and airways and the effect of antioxidants. Life Sci 1999;65:1909±17. [31] Gosling JA, Kung LS, Dixon JS, Horan P, Whitbeck C, Levin RM. Correlation between the structure and function of the urinary bladder following partial outlet obstruction. J Urol 2000;163: 1349±56.
[32] SchroÈder A, Chichester P, Kogan BA, Longhurst PA, Lieb J, Levin, RM. Effect of chronic bladder outlet obstruction on the blood ¯ow of the rabbit urinary bladder. J Urol 165:640±6. [33] Parekh MH, Lobel R, O'Connor LJ, Leggett RE, Levin RM. Protective effect of vitamin E on the response of the rabbit bladder to partial outlet obstruction. J Urol 2001;166:341±6. [34] Levin RM, Das AK, Haugaard N, Novitsky Y, Horan P, Leggett RE, et al. Bene®cial effects of Tadenan therapy following two weeks of partial outlet obstruction in the rabbit. Neurourol Urodyn 1997;16: 583±99. [35] Levin RM, Kawashima Y, Mizutani K, Leggett RE, Whitbeck C, Horan P. Effect of oral Kohki tea on the bladder dysfunctions induced by severe partial outlet obstruction. J Urol 2002;167:2260-6. [36] Das AK, Leggett RE, Whitbeck C, Horan P, Levin RM. Effect of doxazosin on bladder function following partial outlet obstruction in rats. Neurourol Urodyn 2002;21:160±6. [37] Pinggera GM, Schuster A, Pallwein L, Klauser A, Frauscher F, FurtmuÈller B. Alpha-blockers increase vesical and prostatic blood ¯ow and bladder capacity. J Urol 2002;167(4 Suppl):373 [Abstract 1485]. [38] Korstanje C, de Wijn S, Huisman A, Koorn J, van den Hoven W. Tamsulosin and bladder hypertrophy in a rabbit model. Eur Urol Suppl 2002;1(1):61 [Abstract 233].