- Email: [email protected]
MP17-16 MICROSTIMULATION OF HIPPOCAMPUS INHIBITS MICTURITION REFLEX IN URETHANE-ANESTHETIZED RATS
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Saturday, May 17, 2014
efficiency was lowered at 12 weeks and 20 weeks in comparison to the controls. The bladder compliance and capacity of 12 week but not 20 week DKO mice was lower than age-matched controls. An abnormal voiding pattern was observed in the DKO mice as determined by VSOP analysis with lower micturition volumes, and higher frequency of small volume voids. In TKO animal, the voiding parameters were restored to normal levels equivalent to age-matched controls. CONCLUSIONS: Our findings demonstrate that secondary complication of diabetes, DBD, is corrected in diabetic animals with hepatic-specific genetic modification in the TKO mice, with deletions of IRS1/IRS2 & Foxo1 genes. Our findings suggest that the DKO and TKO reversible diabetic mouse model system is a rational model to evaluate the pathophysiology and molecular alterations associated with diabetic cystopathy. Source of Funding: NIH/NIDDK grant (Diabetes Complications Consortium Pilot & Feasibility Project, 09MCG72 and 27532-20) and the American Urological Association/Pfizer Pharmaceutical Company competitive grants to AFO.
MP17-15 ANGIOTENSIN II AS A NOVEL REGULATOR FOR UROTHELIAL AND BLADDER FUNCTION Guiping Sui, London, United Kingdom; Lisa Adjei, Guildford, United Kingdom; Rui Wu*, Birmingham, United Kingdom; Max Robers, Guildford, United Kingdom; Bruce Montgomery, Frimley, United Kingdom; Jian-Mei Li, Changhao Wu, Guildford, United Kingdom INTRODUCTION AND OBJECTIVES: Angiotensin II (AngII) is an endogenous vasoconstrictor and a trophic factor. It has important pathological implications in detrusor hypertrophy, bladder outflow obstruction and oxidative stress. Recent evidence suggests that angiotensin receptors may exist in the urothelium e the newly recognized sensory structure. We examined the role of AngII as a novel regulator for urothelium and bladder function. METHODS: Urothelium-attached and denuded detrusor muscle and urothelial sheets were prepared from guinea-pig bladders. Human bladder mucosas were obtained at cystoscopy with ethical approval. The tissues were superfused in a physiological saline and tied to a tensin transducer to record contractions. A luciferin-luciferase assay measured tissue ATP release. Immunofluorescence microscopy determined AT1 receptor expression. RESULTS: Urothelium-attached but not denuded detrusor muscle strips exhibited un-provoked spontaneous contractions. AngII (200 nM e 1 mM) significantly enhanced urothelium-mediated spontaneous contractions (control: 248120mN/mg tissue; 200nM AngII: 442165mN/mg tissue, 25652% of control, n¼9, p<0.05). AT1 receptor antagonist ZD7155 (1-10mM) suppressed the positive inotropic action of AngII (n¼6). Confocal microscopy identified positive staining for AT1 antibodies throughout the urothelial layer. AngII increased ATP release from the urothelium-smooth muscle preparations (control: 9727pmoles/min/g tissue; 200nM AngII: 325159 pmoles/min/g tissue, 36192% of control; n¼14, p<0.05). The main source of ATP release was from the urothelium. Application of exogenous reactive oxygen species (ROS) - hydrogen peroxide, increased the urotheliumdependent spontaneous muscle contractions (16021% of control, n¼5, p<0.05) while blocking endogenous ROS production by apocynin had the opposite effect. ZD7155 (1mM) alone also attenuated the urothelium-dependent muscle activity (from 16699mN to 11870mN, 733.6% of control, n¼5, p<0.05). AngII generated similar ATP release in human urothelium. CONCLUSIONS: Angiotensin II exerts stimulatory effect on urothelium-dependent detrusor muscle activity mediated by AT1 receptors. Angiotensin II releases ATP from the urothelium to generate a paracrine effect. Reactive oxygen species may confer further action for angiotensin II with pathological implications. Suppression of the muscle
e141
activity by AT1 antagonist without exogenous agonists suggests that endogenous angiotensin II release occurs in the bladder. We propose the angiotensin system as a novel regulator for urothelium and bladder function. Source of Funding: The BBSRC Age UK.
MP17-16 MICROSTIMULATION OF HIPPOCAMPUS INHIBITS MICTURITION REFLEX IN URETHANE-ANESTHETIZED RATS Yosuke Matsuta*, Fukui, Japan; Bing Shen, James Roppolo, William de Groat, Pittsburgh, PA; Osamu Yokoyama, Fukui, Japan; Changfeng Tai, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Hippocampus is known to be involved in emotion and memory functions. Although recent brain imaging studies have indicated the activation of hippocampus during micturition control, there is no report that directly examines the role of hippocampus in micturition reflex. The purpose of this study is to investigate how direct stimulation of hippocampus affects the micturition reflex. METHODS: A total of 8 female Sprague-Dawley rats weighing 300g under urethane anesthesia (1.2 g/kg) were used in this study. The bladder was cannulated through urethra using a PE-50 catheter, and isovolumetric bladder contractions were recorded. After the catheter insertion, rat was fixed on a stereotaxic frame and then the brain was exposed by removing a small piece of skull. A concentric tungsten electrode was inserted stereotaxically into the hippocampus following the coordinates of the rat brain map by Paxinos and Watson. An electrical stimulation (0.2 ms pulse width and 20 V) with frequencies from 1 to 200 Hz was applied to the hippocampus to evaluate its effect on isovolumetric bladder contraction. After the stimulation, the brain was resected and preserved in the formalin for histology. RESULTS: An electrical stimulation of 20 Hz frequency maximally inhibited the isovoliumetric bladder contractions with the microelectrodes in the dorsal areas of hippocampus. The inhibition lasted for a much longer time than the stimulation duration. Intraperitoneal administration of naloxone (1 mg/kg) shortened the duration of inhibition, but it did not completely eliminate the inhibition. The stimulation never induced any excitatory response. CONCLUSIONS: These results indicate that electrical activation of the neurons in hippocampus inhibits the micturition reflex. Understanding the brain function in micturition control is important for the treatments of bladder dysfunctions caused by brain damages or disorders.
THE JOURNAL OF UROLOGYâ
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Vol. 191, No. 4S, Supplement, Saturday, May 17, 2014
LUT SEPs provide promising measures to monitor treatment outcome and functional recovery. Further investigations are warranted in larger cohorts and patients. Source of Funding: Swiss National Science Foundation
MP17-18 BIDIRECTIONAL COMMUNICATION BETWEEN AFFERENT NEURONS AND UROTHELIAL CELLS IN THE MOUSE URINARY BLADDER Youko Ikeda*, Irina Zabbarova, Anthony Kanai, Pittsburgh, PA
Source of Funding: Supported by the NIH under grants DK068566, DK-090006 and DK-091253.
MP17-17 SENSORY EVOKED CORTICAL POTENTIALS OF THE LOWER URINARY TRACT IN HEALTHY MEN Martina D. Liechti, Stephanie Knuepfer*, Flavia Gregorini, Martin Schubert, Armin Curt, Thomas M. Kessler, Ulrich Mehnert, €rich, Switzerland Zu INTRODUCTION AND OBJECTIVES: Sensory evoked cortical potentials (SEPs) are established diagnostic tools to objectively assess sensory function within different areas of the body. Such tool can be of diagnostic value for the assessment of lower urinary tract (LUT) afferent properties. Recently we demonstrated reliable recording of SEPs from different LUT sites in healthy females. However, still little is known from male LUT. Aim of the study was to evaluate the feasibility and reliability of SEP recording following stimulation at different localizations in male LUT. METHODS: Ten healthy men (age:19-37 years, height: 1.850.05 meters) were measured twice (intervall:1-3 weeks). During electroencephalographic (EEG) recording, one-millisecond, repetitive (0.5Hz) square wave stimuli were applied via a special transurethral 8F catheter to urethral (distal, membranous, proximal) and endovesical stimulation sites (trigone, bladder wall). The bladder was drained and refilled with 60mL contrast medium after each stimulation cycle. EEG data were filtered (0.5-30Hz band-pass plus 50Hz notch filter), segmented and averaged per subject and localization. Focus of analysis was cortical component N1 recorded from the Cz electrode referenced to Fz (10/20 system). Statistical tests comprised analysis of variance (ANOVA), intraclass correlation coefficients (ICC), linear regressions, and paired t-tests. Values are given as mean standard deviation. RESULTS: LUT SEPs could be detected with a prominent N1 and P2 and a small P1 component in Cz-Fz signal. N1 latencies were most reliable (ICC:0.61-0.77) and localization-specific (bladder wall:127.118.6ms, trigone:133.514.2ms; urethra: proximal:130.8 17.9ms, membranous:126.132.2ms, distal:113.014.6ms). N1P2 amplitudes were highly reproducible (ICC:0.89-0.96) within subjects, but greatly varied between subjects. N1 latencies resulting from stimulation at the bladder wall, membranous and distal urethra, demonstrated a significant (all p<0.01) negative correlation with subject age. CONCLUSIONS: SEPs can be reliably recorded following electrical stimulation at different LUT sites in men. Variability of N1P2 amplitudes between subjects seems to be higher compared to within subjects, which can be explained by the individual electrical and pain perception. Location specific differences in N1 latencies might reflect different local afferent innervation, i.e. different fiber density and quality.
INTRODUCTION AND OBJECTIVES: It has been widely hypothesized that urothelial cells communicate unidirectionally with afferent terminals to signal changes in the bladder lumen milieu. However, direct interactions between afferents and urothelial cells have not been clearly demonstrated. Our aim was to examine these interactions using pseudorabies virus (PRV) with the genetically-encoded Ca2+ probe, GCaMP to localize its fluorescence in the mouse urinary bladder. METHODS: For dorsal root ganglia (DRG)-PRV injections, adult female C57Bl/10 mice were anesthetized and a hemilaminectomy performed at the L5-L6 vertebrae level to expose L6-S1 DRG. 2 ml of PVR468-GCaMP3 (106 pfu/ml) was slowly injected into each DRG using a 1.0 mm glass micropipette with a 10 mm beveled tip. The muscle and skin were sutured, animals allowed to recover and used for experiments after 2-3 days. For sham controls, sterile saline was injected in place of viral vector. In additional experiments, 10 ml of PRV was injected into the wall of the descending colon or tail muscle. Isolated DRG or whole bladder sheets were fixed, sectioned and examined for GCaMP fluorescence. RESULTS: Following DRG-PRV injections GCaMP labeling was found in afferent nerves in the detrusor (DM) and within the urothelium (UT), but not the lamina propria (LP) (figure 1A). Labeling in the UT appeared punctate or contained inside vesicles, suggesting there may be urothelial uptake of GCaMP or PRV from afferents. In colon and tail injections, a similar GCaMP expression pattern was found compared to DRG injections (B and C). Colon/tail injected animals also had dense expression in L6/S1 DRG, demonstrating that these structures have shared afferent innervation with the bladder (D and E). Fluorescence was not detected in the DRG or bladders of sham control animals (not shown). CONCLUSIONS: The established concept has been that urothelial cells signal to suburothelial afferents to ‘sense’ changes within the bladder lumen milieu. We have demonstrated that communication between afferents and urothelial cells is bidirectional, where afferents can also signal to urothelial cells. Changes in the dynamics of urothelialafferent communication may underlie sensory disorders of the lower urinary tract.
Source of Funding: R01DK071085 to A. Kanai.
Vol. 191, No. 4S, Supplement, Saturday, May 17, 2014
efficiency was lowered at 12 weeks and 20 weeks in comparison to the controls. The bladder compliance and capacity of 12 week but not 20 week DKO mice was lower than age-matched controls. An abnormal voiding pattern was observed in the DKO mice as determined by VSOP analysis with lower micturition volumes, and higher frequency of small volume voids. In TKO animal, the voiding parameters were restored to normal levels equivalent to age-matched controls. CONCLUSIONS: Our findings demonstrate that secondary complication of diabetes, DBD, is corrected in diabetic animals with hepatic-specific genetic modification in the TKO mice, with deletions of IRS1/IRS2 & Foxo1 genes. Our findings suggest that the DKO and TKO reversible diabetic mouse model system is a rational model to evaluate the pathophysiology and molecular alterations associated with diabetic cystopathy. Source of Funding: NIH/NIDDK grant (Diabetes Complications Consortium Pilot & Feasibility Project, 09MCG72 and 27532-20) and the American Urological Association/Pfizer Pharmaceutical Company competitive grants to AFO.
MP17-15 ANGIOTENSIN II AS A NOVEL REGULATOR FOR UROTHELIAL AND BLADDER FUNCTION Guiping Sui, London, United Kingdom; Lisa Adjei, Guildford, United Kingdom; Rui Wu*, Birmingham, United Kingdom; Max Robers, Guildford, United Kingdom; Bruce Montgomery, Frimley, United Kingdom; Jian-Mei Li, Changhao Wu, Guildford, United Kingdom INTRODUCTION AND OBJECTIVES: Angiotensin II (AngII) is an endogenous vasoconstrictor and a trophic factor. It has important pathological implications in detrusor hypertrophy, bladder outflow obstruction and oxidative stress. Recent evidence suggests that angiotensin receptors may exist in the urothelium e the newly recognized sensory structure. We examined the role of AngII as a novel regulator for urothelium and bladder function. METHODS: Urothelium-attached and denuded detrusor muscle and urothelial sheets were prepared from guinea-pig bladders. Human bladder mucosas were obtained at cystoscopy with ethical approval. The tissues were superfused in a physiological saline and tied to a tensin transducer to record contractions. A luciferin-luciferase assay measured tissue ATP release. Immunofluorescence microscopy determined AT1 receptor expression. RESULTS: Urothelium-attached but not denuded detrusor muscle strips exhibited un-provoked spontaneous contractions. AngII (200 nM e 1 mM) significantly enhanced urothelium-mediated spontaneous contractions (control: 248120mN/mg tissue; 200nM AngII: 442165mN/mg tissue, 25652% of control, n¼9, p<0.05). AT1 receptor antagonist ZD7155 (1-10mM) suppressed the positive inotropic action of AngII (n¼6). Confocal microscopy identified positive staining for AT1 antibodies throughout the urothelial layer. AngII increased ATP release from the urothelium-smooth muscle preparations (control: 9727pmoles/min/g tissue; 200nM AngII: 325159 pmoles/min/g tissue, 36192% of control; n¼14, p<0.05). The main source of ATP release was from the urothelium. Application of exogenous reactive oxygen species (ROS) - hydrogen peroxide, increased the urotheliumdependent spontaneous muscle contractions (16021% of control, n¼5, p<0.05) while blocking endogenous ROS production by apocynin had the opposite effect. ZD7155 (1mM) alone also attenuated the urothelium-dependent muscle activity (from 16699mN to 11870mN, 733.6% of control, n¼5, p<0.05). AngII generated similar ATP release in human urothelium. CONCLUSIONS: Angiotensin II exerts stimulatory effect on urothelium-dependent detrusor muscle activity mediated by AT1 receptors. Angiotensin II releases ATP from the urothelium to generate a paracrine effect. Reactive oxygen species may confer further action for angiotensin II with pathological implications. Suppression of the muscle
e141
activity by AT1 antagonist without exogenous agonists suggests that endogenous angiotensin II release occurs in the bladder. We propose the angiotensin system as a novel regulator for urothelium and bladder function. Source of Funding: The BBSRC Age UK.
MP17-16 MICROSTIMULATION OF HIPPOCAMPUS INHIBITS MICTURITION REFLEX IN URETHANE-ANESTHETIZED RATS Yosuke Matsuta*, Fukui, Japan; Bing Shen, James Roppolo, William de Groat, Pittsburgh, PA; Osamu Yokoyama, Fukui, Japan; Changfeng Tai, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Hippocampus is known to be involved in emotion and memory functions. Although recent brain imaging studies have indicated the activation of hippocampus during micturition control, there is no report that directly examines the role of hippocampus in micturition reflex. The purpose of this study is to investigate how direct stimulation of hippocampus affects the micturition reflex. METHODS: A total of 8 female Sprague-Dawley rats weighing 300g under urethane anesthesia (1.2 g/kg) were used in this study. The bladder was cannulated through urethra using a PE-50 catheter, and isovolumetric bladder contractions were recorded. After the catheter insertion, rat was fixed on a stereotaxic frame and then the brain was exposed by removing a small piece of skull. A concentric tungsten electrode was inserted stereotaxically into the hippocampus following the coordinates of the rat brain map by Paxinos and Watson. An electrical stimulation (0.2 ms pulse width and 20 V) with frequencies from 1 to 200 Hz was applied to the hippocampus to evaluate its effect on isovolumetric bladder contraction. After the stimulation, the brain was resected and preserved in the formalin for histology. RESULTS: An electrical stimulation of 20 Hz frequency maximally inhibited the isovoliumetric bladder contractions with the microelectrodes in the dorsal areas of hippocampus. The inhibition lasted for a much longer time than the stimulation duration. Intraperitoneal administration of naloxone (1 mg/kg) shortened the duration of inhibition, but it did not completely eliminate the inhibition. The stimulation never induced any excitatory response. CONCLUSIONS: These results indicate that electrical activation of the neurons in hippocampus inhibits the micturition reflex. Understanding the brain function in micturition control is important for the treatments of bladder dysfunctions caused by brain damages or disorders.
THE JOURNAL OF UROLOGYâ
e142
Vol. 191, No. 4S, Supplement, Saturday, May 17, 2014
LUT SEPs provide promising measures to monitor treatment outcome and functional recovery. Further investigations are warranted in larger cohorts and patients. Source of Funding: Swiss National Science Foundation
MP17-18 BIDIRECTIONAL COMMUNICATION BETWEEN AFFERENT NEURONS AND UROTHELIAL CELLS IN THE MOUSE URINARY BLADDER Youko Ikeda*, Irina Zabbarova, Anthony Kanai, Pittsburgh, PA
Source of Funding: Supported by the NIH under grants DK068566, DK-090006 and DK-091253.
MP17-17 SENSORY EVOKED CORTICAL POTENTIALS OF THE LOWER URINARY TRACT IN HEALTHY MEN Martina D. Liechti, Stephanie Knuepfer*, Flavia Gregorini, Martin Schubert, Armin Curt, Thomas M. Kessler, Ulrich Mehnert, €rich, Switzerland Zu INTRODUCTION AND OBJECTIVES: Sensory evoked cortical potentials (SEPs) are established diagnostic tools to objectively assess sensory function within different areas of the body. Such tool can be of diagnostic value for the assessment of lower urinary tract (LUT) afferent properties. Recently we demonstrated reliable recording of SEPs from different LUT sites in healthy females. However, still little is known from male LUT. Aim of the study was to evaluate the feasibility and reliability of SEP recording following stimulation at different localizations in male LUT. METHODS: Ten healthy men (age:19-37 years, height: 1.850.05 meters) were measured twice (intervall:1-3 weeks). During electroencephalographic (EEG) recording, one-millisecond, repetitive (0.5Hz) square wave stimuli were applied via a special transurethral 8F catheter to urethral (distal, membranous, proximal) and endovesical stimulation sites (trigone, bladder wall). The bladder was drained and refilled with 60mL contrast medium after each stimulation cycle. EEG data were filtered (0.5-30Hz band-pass plus 50Hz notch filter), segmented and averaged per subject and localization. Focus of analysis was cortical component N1 recorded from the Cz electrode referenced to Fz (10/20 system). Statistical tests comprised analysis of variance (ANOVA), intraclass correlation coefficients (ICC), linear regressions, and paired t-tests. Values are given as mean standard deviation. RESULTS: LUT SEPs could be detected with a prominent N1 and P2 and a small P1 component in Cz-Fz signal. N1 latencies were most reliable (ICC:0.61-0.77) and localization-specific (bladder wall:127.118.6ms, trigone:133.514.2ms; urethra: proximal:130.8 17.9ms, membranous:126.132.2ms, distal:113.014.6ms). N1P2 amplitudes were highly reproducible (ICC:0.89-0.96) within subjects, but greatly varied between subjects. N1 latencies resulting from stimulation at the bladder wall, membranous and distal urethra, demonstrated a significant (all p<0.01) negative correlation with subject age. CONCLUSIONS: SEPs can be reliably recorded following electrical stimulation at different LUT sites in men. Variability of N1P2 amplitudes between subjects seems to be higher compared to within subjects, which can be explained by the individual electrical and pain perception. Location specific differences in N1 latencies might reflect different local afferent innervation, i.e. different fiber density and quality.
INTRODUCTION AND OBJECTIVES: It has been widely hypothesized that urothelial cells communicate unidirectionally with afferent terminals to signal changes in the bladder lumen milieu. However, direct interactions between afferents and urothelial cells have not been clearly demonstrated. Our aim was to examine these interactions using pseudorabies virus (PRV) with the genetically-encoded Ca2+ probe, GCaMP to localize its fluorescence in the mouse urinary bladder. METHODS: For dorsal root ganglia (DRG)-PRV injections, adult female C57Bl/10 mice were anesthetized and a hemilaminectomy performed at the L5-L6 vertebrae level to expose L6-S1 DRG. 2 ml of PVR468-GCaMP3 (106 pfu/ml) was slowly injected into each DRG using a 1.0 mm glass micropipette with a 10 mm beveled tip. The muscle and skin were sutured, animals allowed to recover and used for experiments after 2-3 days. For sham controls, sterile saline was injected in place of viral vector. In additional experiments, 10 ml of PRV was injected into the wall of the descending colon or tail muscle. Isolated DRG or whole bladder sheets were fixed, sectioned and examined for GCaMP fluorescence. RESULTS: Following DRG-PRV injections GCaMP labeling was found in afferent nerves in the detrusor (DM) and within the urothelium (UT), but not the lamina propria (LP) (figure 1A). Labeling in the UT appeared punctate or contained inside vesicles, suggesting there may be urothelial uptake of GCaMP or PRV from afferents. In colon and tail injections, a similar GCaMP expression pattern was found compared to DRG injections (B and C). Colon/tail injected animals also had dense expression in L6/S1 DRG, demonstrating that these structures have shared afferent innervation with the bladder (D and E). Fluorescence was not detected in the DRG or bladders of sham control animals (not shown). CONCLUSIONS: The established concept has been that urothelial cells signal to suburothelial afferents to ‘sense’ changes within the bladder lumen milieu. We have demonstrated that communication between afferents and urothelial cells is bidirectional, where afferents can also signal to urothelial cells. Changes in the dynamics of urothelialafferent communication may underlie sensory disorders of the lower urinary tract.
Source of Funding: R01DK071085 to A. Kanai.