MP21-11 CLARITY ILLUMINATES THE STRUCTURAL AND MOLECULAR CHARACTERISTICS OF MOUSE GENITOURINARY TISSUES -- OVER AND OVER AGAIN

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aging male. This study aims to evaluate the cystometric effects of chronic tadalafil administration in castrated mice and the effects of testosterone replacement. METHODS: A total of 42 mice were randomized to six groups. Group 1 (L-NAME): L-name (60mg/kg), which is an oxide-nitric sintethase inhibitor, was administrated in drinking water. Group 2 (DTAD): Lname þ diluent of tadalafil (mannitol and xantane gum). Group 3 (TAD): L-name þ daily tadalafil (4mg/kg). Group 4 (ORQ): L-name þ orchiectomy. Group 5(ORQþTAD): L-name þ orchiectomy þ tadalafil. Group 6 (TEST): L-name þ ORQ þ tadalafil þ testosterone undecanoate SC single dose (100mg/kg). During cystometry, five parameters were measured: number of non-voiding contractions before first micturition (NVC) and micturition frequency (FM). Threshold pressure (TP), basal pressure (BP), and volume threshold (VT) were expressed as mmHg. Statistical analysis were held with GraphPad Prismâ, using analysis of variance (ANOVA) and Tukey’s tests. Level of significance was 5% (P<0.05). Mean and standard desviation (SD) were analysed. RESULTS: TAD (1.281.89), ORQ(0.160.40), and TEST (1.141.21) Groups had lower number of NVC compared to L-name (5.424.11) Group. ORQ þ TAD(1,51,64) and ORQ had similar similar NVC (p¼0,0042). TAD (1.060.29) and ORQ (0.590.20) groups had lower FM compared L-name Group (2.160.50). ORQ þ TAD (0.710.24) and ORQ had similar FM. TEST (2,970,45) had higher FM between groups (p<0,0001). TAD (27.577.41) and ORQ (275.51) Groups had lower TP compared to L-name (41.4310.86). ORQ þ TAD (32.177.27) and ORQ had similar TP. TEST (42.146.09) had higher TP compared to ORQ (275.51) and TAD Groups (p¼0,0006). TAD (18.713.94) and ORQ (1310.94) Groups had lower BP compared to L-name (31.144.56). ORQ(1310.94) and ORQþTAD (19.833.25) had similar BP. TEST(285.83) had higher BP compared to ORQ (p¼0,0006). There were no statistical diferences in volume threshold between groups. There were no diferences between DTAD and L-name in any parameter. CONCLUSIONS: Tadalafil and castration were equally effective reducing detrusor overactivity in mice with nitric oxide chronic deficiency. Co-treatment of testosterone with tadalafil to hipogonadic rats improves frequency of micturition, basal and threshold pressure. Source of Funding: None

MP21-10 PHYSICAL ACTIVITY MAY PREVENT BLADDER DYSFUNCTION IN OBESE RATS THROUGH OVER-EXPRESSION OF INSULIN SIGNALING RELATED GENES Andre Matos Oliveira*, Fernando F. Fonseca, Sabrina T. Reis, Vanessa Guimaraes, Katia R. Leite, William C. Nahas, Miguel Srougi, Alberto A. Antunes, Sao Paulo, Brazil INTRODUCTION AND OBJECTIVES: Sedentarism and obesity have been reported as relevant risk factors for the development of bladder dysfunction through alterations in the insulin-signaling pathway. In the present study, we investigated the role of physical activity in bladder insulin-signaling pathway in normal fed and high fat fed rats. METHODS: Twenty eight male Wistar rats with eight weeks old were used in this experiment and were randomly divided into four groups: 1. physical activity (PA) and normal diet; 2. PA and high fat diet; 3. sedentary (S) and normal diet; 4. S and high fat diet. Food and water were provided ad libitum. Groups 1 and 2 were submitted to a swimming training protocol for 10 weeks, executed five times a week with duration of 60 minutes per day. At the end of the protocol, bladders were dissected and stored. We measured bladder gene expression levels of protein kinase A and B (PKCa and PKCb), insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), endothelial nitric oxide synthase (eNOS), protein kinase B (AKT) and tribbles homolog 3

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(Trib3) e AKT supressor - through the quantitative real-time polymerase chain reaction (qRT-PCR) method. RESULTS: After 10 weeks, groups 1 and 2 had less visceral fat when compared to the groups 3 (29.4 vs 37.9 grams; p<0.05) and 4 (31.8 vs 41.9 grams; p<0.05), respectively. When comparing normal fed rats (group 1 vs group 3; Graph 1), IRS2, eNOS, AKT, PKCa, PKCb genes were more over-expressed in bladder of rats submitted to PA (median 1.49, 2.28, 1.47, 1.95 and 1.1 times respectively) and IRS1 and Trib3 were more under-expressed (median 0.63 and 0.37 times respectively). Comparing high fat fed rats (group 2 vs group 4; Graph 2) we found overexpression in IRS1, IRS2, eNOS, AKT, PKCa and PKCb (median 9.27, 3.27, 7.50, 6.22, 2.63, 5.23 times respectively) and Trib3 did not change the pattern of expression (median 1.08). CONCLUSIONS: PA improved intracellular bladder insulinsignaling pathway, with more pronounced results in obese rats. These findings suggest a protective effect of PA on obesity-related bladder dysfunctions.

Source of Funding: FAPESP - protocol 2014/08368-0

MP21-11 CLARITY ILLUMINATES THE STRUCTURAL AND MOLECULAR CHARACTERISTICS OF MOUSE GENITOURINARY TISSUES – OVER AND OVER AGAIN Yan Liu, Herbert Lepor, Xue-Ru Wu, Ellen Shapiro*, New York, NY INTRODUCTION AND OBJECTIVES: CLARITY (Nature, 2013, 497:332-7) is a new technique that chemically transforms intact tissue into an optically transparent and permeable hydrogel-hybridized form, replacing lipid bilayers while preserving fine structure and biomolecules including proteins and nucleic acids. This technique is advantageous since human genitourinary (GU) tissues, normal and pathological, are not readily available. With CLARITY, the same specimen can be used for repetitive immunostaining and high resolution 3-D imaging. We report the first application of CLARITY using mouse GU tissues. METHODS: Bladder, prostate and seminal vesicle of adult male mice were perfused in hydrogel, embedded in polymerized hydrogel and clarified by circulating clearing buffer. Clarified tissues were incubated with primary and secondary antibodies and imaged by confocal microscopy with water-immersion objective. To strip primary antibodies, samples were incubated in clearing buffer and de-stained tissues were re-stained with different antibodies (bladder with anti-S100 and then anti-cytokeratin (34bE12) and anti-uroplakin Ia; prostate with antiandrogen receptor (AR); seminal vesicle with (34bE12). RESULTS: CLARITY successfully transformed intact mouse bladder, prostate and seminal vesicle into optically transparent tissues. To attain detailed, global structure and molecular information of bladder nerve, clarified bladder was stained by anti-S100 antibody. High resolution imaging and 3D reconstruction showed network distribution of nerve structures within well preserved structures of the entire bladder. Imaging of clarified, anti-AR-stained prostate clearly showed AR

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distribution in an intact prostate. 34bE12 staining of clarified seminal vesicle prominently labeled all the basal cells. To explore the feasibility of multi-rounds of molecule phenotyping, anti-S100 was stripped from the bladder and anti-UPIa and 34beta E12 were used for re-staining. Anti-UPIa specifically labeled the superficial umbrella cells, whereas 34bE12 stained the basal cells of the urothelium. CLARITY preserved the fine structure of all GU samples tested and multiple rounds of molecular phenotyping did not cause any tissue damage. CONCLUSIONS: This is the first report of the use of CLARITY in mouse GU tissues. Our preliminary work shows that CLARITY has the ability to analyze GU organs, repetitively with multiple antibodies which can be compared to molecular data to further our understanding of normal and pathological states. Source of Funding: none

MP21-12 TREATMENT OF RADIATION CYSTITIS VIA P75 RECEPTOR BLOCKADE Youko Ikeda*, Irina Zabbarova, Kathryn Lemon, Neil Lamarre, Michael Epperly, Anthony Kanai, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Ionizing radiation such as that used for treatment of pelvic malignancies can induce cystitis and potentially long-term consequences on bladder function. The urothelial cell layer is highly radiosensitive and thought to be the principal site of cellular damage in the bladder. The proneurotrophin specific p75 receptor is one of the key initiators of the apoptotic cascade; and its blockade may be a possible therapeutic option for prevention and treatment of urothelial damage. Therefore, in this study we examined the effect of an orally administrable p75 receptor blocker, LM11A-31, on irradiation-induced bladder overactivity. METHODS: Adult female C57BL/6 mice were used in this study. Mice were anesthetized (Avertin, 5 mg/kg), a lower midline incision was made and the urinary bladder was externalized for selective irradiation by a linear accelerator (10 Gray). LM11A-31 was administered by oral gavage (100 mg/kg in water) starting one day before irradiation and continued daily until 7 days post-irradiation. Bladder function was evaluated by decerebrate voiding cystometry. RESULTS: Following bladder irradiation, cystometries demonstrated decreased intercontractile intervals and decreased bladder compliance compared to control mice (Figure 1A and B). Daily LM11A31 administration prevented the development of irradiation-induced overactivity (1C). CONCLUSIONS: Urothelial apoptosis is a major consequence of ionizing radiation that leads to the development of radiation cystitis. We have demonstrated in this study that radiation-induced bladder overactivity is ameliorated by p75 receptor inhibition. Accordingly, p75 receptor antagonists could be potential therapeutic agents for prevention of radiation cystitis and as a treatment modality for other conditions associated with compromised urothelial integrity (e.g., interstitial cystitis).

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MP21-13 FUNCTIONAL ROLES OF METABOTROPIC GLUTAMATE RECEPTOR SUBTYPE 1A IN CONTROL OF LOWER URINARY TRACT ACTIVITY IN MICE Mitsuharu Yoshiyama*, Tsutomu Mochizuki, Masayuki Takeda, Chuo, Japan INTRODUCTION AND OBJECTIVES: Glutamic acids have been implicated in control of lower urinary tract (LUT) activity. Glutamate receptors are constituted of two major classes: the ionotropic glutamate receptors and the metabotropic glutamate receptors (mGluRs). This study was conducted using mice lacking mGluR1a, one of the mGluR subtypes, to determine if neural transmission via mGluR1a is involved in a mouse LUT function. METHODS: Experiments were performed on female mGluR1a knockout (KO) mice and their wild type (WT) littermates (C57BL/6, 12e14 week-old). For evaluating micturition behavior, conscious mice were individually placed in metabolism cages, and frequency-volume charts (FVCs) were measured. For assessing reflex micturition, mice were decerebrated under sevoflurane anesthesia and CMG recordings were conducted under unanesthetized conditions by continuously infusing saline (10 ml/min). Evaluated parameters are: water intake, voided urine volume, frequency of void, and urine volume/void for metabolism cage study; and bladder compliance (BCP), volume threshold for inducing micturition (VT), voiding efficiency (VE), and maximal voiding pressure (MVP) for CMG study. All values are expressed as mean  S.E.M. RESULTS: FVC measurements exhibited that KO mice voided less frequently and released larger volume/void than WT mice (Fig. 1a). No differences were found between KO and WT in water intake and voided urine volume per day. CMG studies revealed that significant differences between KO and WT were found not only in storage parameters (i.e., BCP and VT) but also in voiding parameters (i.e., VE and MVP) (Fig. 1b). CONCLUSIONS: Increases of volume/void in FVC and of VT and BCP in CMG in KO mice imply that neural transmission via mGluR1a is involved in afferent excitatory transmission in storage phase. Lower VE and higher MVP in KO, compared to WT, may be due to higher urethral resistance in KO mice, thereby suggesting the possibility that mGluR1a mechanism also participates in the inhibitory control of external urethral sphincter activity during voiding.

Source of Funding: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) No. 25462507 (to M. Yoshiyama)

Source of Funding: NIH/NIDDK, R01-DK071085 to A. Kanai