e140 THE JOURNAL OF UROLOGY� Vol. 191, No. 4S, Supplement, Saturday, May 17, 2014

vs intact), and 112.8+/-6.4 in SCI rats receiving BBG (p<0.01 vs SCIrats). Cystometric studies showed a higher frequency of non-voidingcontractions in SCI than in BBG-treated SCI rats.

CONCLUSIONS: Inhibition of P2X7R at the SCI site maybe a therapeutic target to improve bladder function by promotingneuroregeneration and decreasing urine NGF, a biomarker for neuro-genic bladder.

Source of Funding: The Houston Methodist Foundation & TheBrown Foundation

MP17-12EFFECTS OF UROTHELIAL P2X3 RECEPTOR INHIBITION ON RATBLADDER CONTRACTILITY

Andrew Ferguson, Broderick Sutton, Timothy B. Boone, Houston, TX;Anthony P. Ford, San Mateo, CA; Alvaro Munoz*, Houston, TX

INTRODUCTION AND OBJECTIVES: Bladder ATP activatesP2X1R in detrusor cells for purinergic contractions and P2X3R in uro-thelium, lamina propria cells and afferent nerves for sensory trans-mission. Using a selective P2X3 receptor antagonist we assessed therole of urothelial P2X3R in the contractile responsiveness of intact orurothelium free bladders.

METHODS: Female Sprague-Dawley rats were intravesicallyinfused with saline or protamine (PR; 10 mg/ml for 30 min). Opencystometry with saline followed by infusion of AF-353 (P2X3 antagonist;10 uM) was performed in urethane anesthetized rats. Bladder pressureduring voiding (BPP), inter-contractile interval (ICI) and bladdercompliance (BC) were calculated. Bladder wash was collected for ATPdetermination (luciferase). Electrical field stimulations (EFS; 100V/1ms/32Hz/10s every 3 min) were applied in longitudinal strips duringsequential application of AF-353, abMeATP (10 uM) and atropine(1uM). Contractile response to KCl (140 mM) was used for normaliza-tion. Timing and amplitude of purinergic contractions were calculated inresponse to abMeATP in different AF-353 concentrations. Histologywas done with H&E and IHC against P2X3 (Abcam, ab90905; 1:2000).Data were analyzed with ANOVA or t-tests with p<0.05 statisticallysignificant.

RESULTS: P2X3Rs are expressed in urothelial and laminapropria cells, with PR treatment removing most urothelial cells. Intra-vesical AF-353 (10uM) reduced BPP (25.5%), and increased both ICI(46.0%) and BC (100%) in control but not in PR rats. Inhibition ofP2X3R during CMG produced a 4.6 fold increase in ATP from controls,without affecting high values in PR animals (310+/-66 vs 432+/-97 pmol/void, saline vs AF-353). KCl contractions were not affected by P2X3inhibition or urothelium removal. Inhibition of P2X3R prevents desen-sitization to abMeATP in intact but not PR strips during EFS. AF-353(10nM or 10uM) increased the response time to abMeATP (30%)independently of urothelial integrity. Desensitization of purinergic con-tractions was prevented when P2X3R were blocked in urothelial cells.

CONCLUSIONS: P2X3R in urothelium and lamina propria mayplay a role in regulating the intravesical release of ATP as well aspreventing P2X1R desensitization. Understanding the role of P2X3R inmodulating contractile and sensory mechanisms would help to designmore effective therapies for bladder dysfunction.

Source of Funding: The Houston Methodist Foundation & TheBrown Foundation

MP17-13FUNCTIONAL-MRI DURING BLADDER CYSTOMETRY IN SPINALCORD-INJURED AND INTACT FEMALE RATS

Kelvin Wong, Timothy B. Boone, Alvaro Munoz*, Houston, TX

INTRODUCTION AND OBJECTIVES: The afferent arm of themicturition reflex is initiated by sensory signals in the bladder andtransmitted to brain centers for storage & release of urine. Our objective

was to determine brain activation using functional-magnetic resonanceimaging (fMRI) in intact and spinal cord injured (SCI) rats during iso-volumetric bladder contractions (IBC).

METHODS: Female Sprague-Dawley rats with SCI (completeT8/T9 transection) were evaluated after 28 days. Urethane-anes-thetized rats had isovolumetric cystometry (CMG) during fMRI viaa suprapubic catheter & closed urethra. Saline infusion stopped at100 cmH2O (cmw). CMG data were analyzed with t-test, expressed asmean+/-SEM, and p<0.05 statistically significant. Simultaneous fMRIwas conducted with a 9.4T Bruker MRI system using a 4-elementsreceiver array and a quadrature volume transmit coil. 2D gradient echo-planar imaging was used to evaluate blood oxygenation level depen-dent (BOLD) responses in the brain during IBC and compared to theempty bladder. Group analysis was conducted based on mixed effectanalysis with FMRIB Software Library v6.0 (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) and a cluster threshold of Z>2. 5 with significance thresholdof p¼0.05.

RESULTS: Isovolumetric pressure was 42+/-4 cmw in intact(n¼7) and 43+/-9 cmw in SCI rats (n¼4). IBC amplitude was 57+/-4 inintact and 5+/-0.5 cmw in SCI rats (p<0.001). Intercontractile intervalwas 99+/-16 s in control and 17+/-3 s in SCI rats (p<0.01). In intact rats(n¼6), mixed effect analysis of IBC showed clustered brain activation inhippocampus, ectorhinal cortex, dentate gyrus, thalamus nucleus,septal nucleus, primary/secondary motor cortex, primary somatosen-sory cortex as well as in the periaqueductal gray matter. SCI rats did notexceed the Z-threshold during IBC. Motion contaminated data wereidentify and excluded as pseudo brain activation due to correlatedIBC motion.

CONCLUSIONS: We standardized a suitable protocol to studysupraspinal activation during reflexive micturition using fMRI. Smallcontractions in SCI rats may represent neurogenic detrusor overactivitycaused by increased excitability of afferent pathways without brainactivation.

Source of Funding: The Houston Methodist Foundation & TheBrown Foundation

MP17-14CORRECTION OF HYPERGLYCEMIA AND HYPERINSULINEMIABY GENETIC MODIFICATION RESTORES BLADDERDYSFUNCTION ASSOCIATED WITH TYPE 2 DIABETES

Zongwei Wang, Vivian Cristofaro, Boston, MA; Zhiyong Cheng,Blacksburg, VA; Hongying Cao, Evgeniy Kreydin, Joseph Gabrielsen,Rongbin Ge, Shulin Wu, Chao Cai, Peng Wu, Maryrose Sullivan,Morris White, Aria Olumi*, Boston, MA

INTRODUCTION AND OBJECTIVES: Diabetes bladderdysfunction (DBD) is a major urologic complication associated with type2 diabetes (DM2). In a mouse model that develops DM2 using a he-patic-specific insulin receptor substrate 1 and 2 (Irs1/Irs2) deletions(double knockout: DKO), we have shown specific molecular alterationsassociated with DBD. Previously, we have shown that DBD occursprogressively with hyperactivity in the early stage and hypoactivity inlate stage of DBD. Here we demonstrate that correction of hypergly-cemia/hyperinsulinemia with a third hepatic-specific deletion of Foxo1gene (i.e.: IRS1/IRS2/Foxo1 triple knockout: TKO) restores the bladderdysfunction in DKO type 2 diabetic animals.

METHODS: Insulin and glucose levels were measured andglucose tolerance tests (GTT) were carried out in DKO, TKO and controlanimals at 4, 8, 12, and 20 weeks of age. Bladder functional alterationswere evaluated by in vivo cystometry and voiding stain on paper(VSOP) for 12 and 20 week old mice. Bladders were harvested for exvivo muscle strip contraction analyses.

RESULTS: The DKO mice developed significant insulinresistance and glucose tolerance starting from 5 weeks of age, and per-sisted at the age of 8, 12, and 20 weeks. TKO animals with hepatic-specific deletion of Irs1/Irs2/Foxo1 genes did not develop hyperglycemiaor hyperinsulinemia. In the diabetic DKO animals, the post void residual(PVR) urine volume in in-vivo cystometry was elevated and the voiding