Murine bladder wall biomechanics following partial bladder obstruction

Evaluation of bladder wall mechanical behavior is important in understanding the functional changes that occur in response to pathologic processes such as partial bladder outlet obstruction (pBOO). In the murine model, the traditional approach of cystometry to describe bladder compliance can prove difficult secondary to small bladder capacity and surgical exposure of the bladder. Here, we explore an alternative technique to characterize murine mechanical properties by applying biaxial mechanical stretch to murine bladders that had undergone pBOO. 5–6 week old female C57/Bl6 mice were ovariectomized and subjected to pBOO via an open surgical urethral ligation and sacrificed after 4 weeks (n=12). Age matched controls (n=6) were also analyzed. Bladders were separated based on phenotype of fibrotic (n=6) or distended (n=6) at the time of harvest. Biaxial testing was performed in modified Kreb's solution at 37 °C. Tissue was preconditioned to 10 cycles and mechanical response was evaluated by comparing axial strain at 50 kPa. The normal murine bladders exhibited anisotropy and were stiffer in the longitudinal direction. All mice showed a loss of anisotropy after 4 weeks of pBOO. The two phenotypes observed after pBOO, fibrotic and distended, exhibited less and more extensibility, respectively. These proof-of-principle data demonstrate that pBOO creates quantifiable changes in the mechanics of the murine bladder that can be effectively quantified with biaxial testing.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.     

Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKalpha and ROKbeta). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.     

Basement membrane components of bronchial epithelium in humans suffering from chronic nonspecific lung diseases

Collagen IV and laminin are important constituents of the basement membrane (BM). By use of immunocytochemistry we examined the occurrence and distribution of these two components in the BM beneath normal, mucoid and metaplastic epithelium of large bronchi in 22 adults suffering from chronic nonspecific lung diseases. Both collagen IV and laminin were expressed as a thin and continuous layer beneath the epithelium in most tissue specimens with normal epithelium. In a few specimens the layer showed interruptions with a patchy distribution of the immunoreactivity. Three patterns of distribution of BM components were found under the metaplastic epithelium. Total absence of immunoreactive collagen IV and laminin was the most common variant. Weak and scarce staining for both proteins in the BM characterized the second pattern. The third variant showed strong collagen IV immunoreactivity but lack of laminin. The BM beneath the mucoid epithelium was characterized by irregular distribution of collagen IV and laminin. We suggest that the occurrence and distributional pattern of the BM components are related to the type of overlying epithelium and connected with an altered synthesis of these components.     

Vascular response of the rabbit bladder to chronic partial outlet obstruction

Partial outlet obstruction of the rabbit urinary bladder results, initially, in a rapid increase in bladder mass and remodeling of the bladder wall. Previously, it was shown that this response was characterized by serosal growth (thickening) which was apparent after 1 day of obstruction, before any visible vascularization was observed. After 1 week of obstruction, significant microvessel formation was seen in the transition region between the detrusor smooth muscle and the thickening serosa; after 2 weeks the entire serosa was vascularized.In this study we investigated the effect of chronic (4 week) partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium. Transverse sections of bladder wall were examined after 4 weeks of no surgery, sham surgery or partial obstruction.The microvessel density of the obstructed rabbit bladder mucosa and detrusor smooth muscle increased relative to augmentation of these compartments while new vessels appeared in the thickening serosa. Although vessel density did not change with obstruction a significant shift in mean vessel circumference to the left occurred indicating a significant increase in the number of microvessels and small vessels consistent with angiogenesis.     

Alterations to network of NO/cGMP-responsive interstitial cells induced by outlet obstruction in guinea-pig bladder

Interstitial cells (ICs) play a role in regulating normal bladder activity. This study explores the possibility that the sub-urothelial and muscle networks of NO/cGMP-responsive ICs are altered in animals with surgically induced outflow obstruction. In sham-operated animals, the urothelium comprised NO-stimulated cGMP-positive (cGMP⁺) umbrella cells, an intermediate layer and a basal layer that stained for nNOS. cGMP⁺ sub-urothelial interstitial cells (su-ICs) were found below the urothelium. cGMP⁺ cells were also associated with the outer muscle layers: on the serosal surface, on the surface of the muscle bundles and within the muscle bundles. Several differences were noted in tissues from obstructed animals: (1) the number of cGMP⁺ umbrella cells and intensity of staining was reduced; (2) the intermediate layer of the urothelium consisted of multiple cell layers; (3) the su-IC layer was increased, with cells dispersed being throughout the lamina propria; (4) cGMP⁺ cells were found within the inner muscle layer forming nodes between the muscle bundles; (5) the number of cells forming the muscle coat (serosa) was increased; (6) an extensive network of cGMP⁺ cells penetrated the muscle bundles; (7) cGMP⁺ cells surrounded the muscle bundles and nodes of ICs were apparent, these nodes being associated with nerve fibres; (8) nerves were found in the lamina propria but rarely associated with the urothelium. Thus, changes occur in the networks of ICs following bladder outflow obstruction. These changes must have functional consequences, some of which are discussed.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni²⁺-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litreE. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search forras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidateras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identifyras binding proteins present in cellular extracts.Abbreviations his-ras histidine-tagged vHa-ras - Ni-NTA Ni²⁺ nitriloacetic acid agarose - IPTG isopropyl thio--D-galactoside     

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.     

Matrigel: history/background,uses, and future applications

Basement membranes are thin sheets of extracellular matrix with many diverse roles in the body. Those in normal tissue are also highly insoluble and resist attempts to extract and characterize their components. A mouse tumor, the EHS tumor, has provided large amounts of basement membrane material, which has facilitated the structural and functional characterization of its components. An extract of the tumor, known as Matrigel, contains components which reconstitute into a solid gel at 37°. This solid basement membrane matrix has been used in both cell culture and in vivo. Matrigel has been utilized in some 12,000-plus publications for a variety of studies with embryonic, normal, and stem or malignant cells. Evidence presented in this Commentary suggests that Matrigel isolated from tumors grown in diverse hosts could exert unique effects that could be helpful in analyzing the causes of various pathologies and for screening possible therapeutic agents.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

The effect of partial outlet obstruction on prostaglandin generation in the rabbit urinary bladder

Partial outlet obstruction of the urinary bladder has been demonstrated to induce specific dysfunctions in cellular and sub-cellular membrane structures within the bladder's smooth muscle and mucosal compartments. Recent studies have linked these membrane dysfunctions to alterations in phospholipid metabolism leading to mobilization of free arachidonic acid, the precursor for synthesis of prostaglandins (PG). The purpose of this study was to determine if partial outlet obstruction of the urinary bladder induces changes in the capacity of bladder smooth muscle and mucosa to generate PG. PG were isolated from control and partially obstructed urinary bladder smooth muscle and mucosa of male New Zealand White (NZW) rabbits. PG concentrations (PGE2, PGF2alpha and PGI2, as its stable metabolite 6-keto-PGF1alpha) were determined after 30 minute incubations using enzyme-linked immunoassay (ELISA) kits. In both control and obstructed rabbit urinary bladders, PG generation was significantly higher in isolated mucosa than muscle tissues. A significantly higher concentration of PGF2alpha, and 6-keto-PGF1alpha was measured in obstructed muscle tissue relative to controls. The concentration of 6-keto-PGF1alpha was also significantly higher than the concentrations measured for PGE2 and PGF2alpha in both control and obstructed smooth muscle samples. The generation of PGE2 was significantly higher in rabbit urinary bladder mucosa than either PGF2alpha or 6-keto-PGF1alpha in both control and obstructed samples. The capacity of obstructed mucosal tissue to generate 6-keto-PGF1alpha was significantly higher than control tissue, while no significant differences in PGE or PGF2alpha generation were noted. These data suggest obstruction of the urinary bladder induce specific elevations in PG in both smooth muscle and mucosal tissues.     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

Adjustable passive stiffness in mouse bladder: regulated by Rho kinase and elevated following partial bladder outlet obstruction

Detrusor smooth muscle (DSM) contributes to bladder wall tension during filling, and bladder wall deformation affects the signaling system that leads to urgency. The length-passive tension (L-T(p)) relationship in rabbit DSM can adapt with length changes over time and exhibits adjustable passive stiffness (APS) characterized by a L-T(p) curve that is a function of both activation and strain history. Muscle activation with KCl, carbachol (CCh), or prostaglandin E(2) at short muscle lengths can increase APS that is revealed by elevated pseudo-steady-state T(p) at longer lengths compared with prior T(p) measurements at those lengths, and APS generation is inhibited by the Rho Kinase (ROCK) inhibitor H-1152. In the current study, mouse bladder strips exhibited both KCl- and CCh-induced APS. Whole mouse bladders demonstrated APS which was measured as an increase in pressure during passive filling in calcium-free solution following CCh precontraction compared with pressure during filling without precontraction. In addition, CCh-induced APS in whole mouse bladder was inhibited by H-1152, indicating that ROCK activity may regulate bladder compliance during filling. Furthermore, APS in whole mouse bladder was elevated 2 wk after partial bladder outlet obstruction, suggesting that APS may be relevant in diseases affecting bladder mechanics. The presence of APS in mouse bladder will permit future studies of APS regulatory pathways and potential alterations of APS in disease models using knockout transgenetic mice.     

雄性大鼠膀胱出口梗阻两种模型制作方法的比较研究

目的 通过采用耻骨后膀胱颈和会阴途径球部尿道部分结扎两种方法,建立雄性大鼠膀胱出口部分梗阻（paritial bladder outlet obstraction,pBOO）模型,并对所建模型进行鉴定和比较,为pBOO后膀胱重构（bladder reconstruction）的深入研究提供一种成活率高,复制性和稳定性较好的动物模型.方法 80只健康雄性Wistar大鼠随机分为三组：I组为假手术组（对照组）,20只;II组为耻骨后途径膀胱颈部分结扎组,30只;III组为会阴途径球部尿道部分结扎组,30只.依据梗阻时间分别将I组、II组、III组大鼠随机分为2周组和4周组,于术后2周和4周对大鼠行尿动力学检测后,完整切除膀胱测其重量,将精囊腺组织和部分膀胱用4%甲醛固定,HE染色观察组织学变化.结果 II组和III组成活率分别为73.3%和80.0%,二者无统计学意义;I组、II组、III组建模手术时间分别为（9.75±2.29）、（17.33±3.54）、（10.77±2.44）min,II组与I组和III组比较差异均有统计学意义;I组、II组、III组的2周组和4周组逼尿肌漏尿点压（detrusor leak point pressure,DLPP）分别为（26.31±2.32）、（27.34±3.93）、（24.68±2.39）mmHg和（26..42±2.41）、（34.23±3.01）、（32.63±3.20）mmHg,I组与II组和III组的4周组比较差异有统计学意义,II组和III组的2周组和4周组比较差异均有统计学意义.结论 耻骨后途经膀胱颈部分结扎和会阴途径球部尿道部分结扎两种方法都能成功建立雄性大鼠pBOO模型,与耻骨后途径相比,会阴途径成活率高,手术操作时间短,复制性和稳定性好.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)