11:00 a.m.-5:30 p.m.
Registration/Information Desk Open
Location: Austin Grand Ballroom Foyer, 6th Floor
11:00 a.m.-5:00 p.m.
Speaker Ready Room Hours
Location: Room 602, 6th Floor
1:00 p.m. - 2:30 p.m.
Panel 1: New Frontiers in Bladder Innervation
Michel Arthur Pontari, MD
New Preclinical Models of Neurogenic Bladder Function
Anna Malykhina, PhD
Dog Model of Nerve Re-Routing for Neurogenic Bladder
Michael R. Ruggieri, Sr., PhD
Translational Aspects of Research Into Neurogenic Bladder
Kenneth M. Peters, MD
2:30 p.m. - 3:30 p.m.
Keynote Lecture: Detrusor Interstitial Cells, The Mystery Resolved
Kenton M. Sanders, PhD
3:30 p.m. - 3:45 p.m.
3:45 p.m. - 5:10 p.m.
Panel 2: ATP Signaling in the Lower Urinary Tract
Vivian Cristofaro, PhD
How Do We Void, the Role of Purinergic Signaling in the Bladder
Chris Fry, BSc, PhD
The Many Faces of Purinergic Regulation in Smooth Muscle: Lessons from the Gut and Bladder
Violeta Mutafova-Yambolieva, MD, PhD
To Void or Not to Void: ATP Signaling in Bladder Mechanotransduction
Sylvia Suadicani, PhD
5:10 p.m. - 5:25 p.m.
5:25 p.m. - 7:40 p.m.
* Basic Science Poster Session I
Matthew O. Fraser, PhD
H. Henry Lai, MD
|Poster #BS1|CHARACTERIZATION OF BACTERIA IDENTIFIED ON EXPLANTED MESH SLINGS USING NEXT-GENERATION SEQUENCING TECHNIQUES
A. Lenore Ackerman MD, PhD, Victoria Scott MD, Guo Liu PhD, Wenyuan Shi PhD and Shlomo Raz MD
Los Angeles, CA
Presented by: A. Lenore Ackerman
Vaginal mesh sling kits for stress urinary incontinence are associated with several severe complications, including mesh extrusion, urinary problems, organ perforation, and vaginal scarring. While most complications are attributable to local pathology (e.g. erosion) or device misplacement (e.g. nerve entrapment), we have observed the delayed development of severe chronic pelvic pain, despite an apparently well-placed graft. We investigated the relationship between alterations in the mesh-associated microbiota with pain symptomatology and local inflammation
Sling mesh segments were isolated from three groups of patients: 1) those with delayed-onset chronic pain, 2) those presenting mesh extrusions >1 cm, and 3) those with isolated urinary retention without pain. Explanted mesh segments were examined by culture-independent, species-specific PCR, denaturing gradient gel electrophoresis, and next-generation deep sequencing methods to detect bacterial communities on explanted mesh segments.
Sixty two patients were enrolled in this study. Exposed mesh segments were uniformly colonized with bacteria mirroring the vaginal microbiome. Unexpectedly, we detected bacterial species on explanted mesh from the group of patients with delayed-onset pain without mesh exposure that differed greatly from the vaginal species present at explantation, suggesting an enrichment of specific pathogenic species able to survive on foreign bodies. This bacterial colonization was absent from patients without pain who underwent mesh removal to treat urinary retention.
We found numerous, distinct bacterial species present on the mesh removed from most subjects in group 1 that were not present in groups 2 and 3 nor in the vaginal microbiome. This suggests the need for further research to explore the relationship of these species and the associated subclinical infection and/or inflammation to delayed-onset chronic pain.
|Poster #BS2|NEUROANATOMICAL EVALUATION OF PERI-VESICAL NERVE PLEXUS IN FEMALE WITH 3T-MR DIFFUSION TENSOR IMAGING
Bilal Farhan MD¹, Hon J. Yu, BSc PhD², Mohammad Helmy MD³ and Gamal Ghoniem MD FACS⁴
¹University of California, Irvine, CA; ²UC, Irivne; ³UC, Irvine; ⁴UC, Irivine
Presented by: Bilal Farhan
OBJECTIVE: To evaluate if tractography or fiber-tracking based on Diffusion Tensor Imaging (DTI) can improve the visualization of peri-vesical nerve fibers. Understanding the exact location and distribution of the entire nerve fibers plexus around the bladder could facilitate newer paradigms and approaches of targeted treatment of bladder dysfunction.
MATERIAL AND METHODS: Pelvic DTI was performed as an add-on to our existing clinical pelvic MRI protocol on a 3Tesla MRI scanner (Siemens, Erlangen, Germany). It was acquired in axial orientation using body- and spine-matrix receiver coils and following acquisition parameters: TR/TE=9500/104 [ms], 30 directions, b-values=0, 600 [s/mm2], and 2 x 2 x 3 mm3 voxel-size. A high-resolution 3d-T2 scan of pelvis (0.9 x 0.9 x 1.0 mm3) in axial orientation was also added to serve as the anatomical template for tractography. DTI was processed using a dedicated workstation (Dynasuite Neuro 2.0, In Vivo Corp, Gainesville, USA) for fiber-tracking/visualization based on manual placement of region-of-interest (ROI). Placement of multiple ROIs in different orientations using anatomical landmarks and color-coded fractional anisotropy (FA) map were carried out by urology fellow (BF) experienced in use of the workstation for visualization of the nerve fibers around the bladder.
RESULTS: DTI showed description of peri-vesical plexus nerve fibers in all directions, while 2D and 3D T2 morphological sequences depicted part of the fibers, in a plane by plane analysis of fiber courses. DTI demonstrated in female patient the spreading of nerve fibers around the bladder. This distribution was on the posterior and lateral bladder walls, and around the bladder neck.
CONCLUSION: This pilot study helps to detect the distribution of peri-vesical nerve fibers plexus, which will help in the targeted therapy for bladder disease including overactive bladder.
|Poster #BS3|ANALYSIS OF VERSICAN AND HYALURONAN DEPOSITION IN THE FIBROTIC AND INFLAMMATORY RESPONSE TO POLYPROPYLENE MESH IN SYMPTOMATIC WOMEN UNDERGOING PELVIC FLOOR MESH REMOVAL
Katherine Amin MD¹, Sarah Adelstein MD¹, Stephen P Evanko PhD², Alvaro Lucioni MD¹, Kathleen Kobashi MD¹, Thomas Wight PhD² and Una Lee MD¹
¹Virginia Mason Medical Center, Department of Urology, Seattle, WA; ²Virginia Mason Medical Center, Benaroya Research Institute Wilske Translational Research, Seattle, WA
Presented by: Katherine Amin
Introduction: Current management of stress urinary incontinence (SUI) and pelvic organ prolapse (POP) includes use of polypropylene mesh. Pathophysiology of mesh complications is not fully elucidated, but fibrosis and inflammation have been identified. Abnormal extracellular matrix (ECM) is associated with (a/w) SUI and POP and in tissues of mesh complications. Proteoglycans, versican and hyaluronan (HA) play a key role in the regulation of ECM. We characterize the presence and distribution of versican and HA in vaginal tissue of women with mesh complications.
Methods: Tissue specimens of mesh removal procedures between 2011-2012 for symptomatic complications were examined by microscopy (H&E) and immunohistochemical staining for versican, (hyaluronan-binding protein a/w inflammation and elastin breakdown), hyaluronan (HA), and α-SMA (smooth muscle protein a/w inflammation and myofibroblast marker). Comparison was performed to control specimens obtained from excess tissue of POP or SUI surgery, with IRB-approval.
Results: Explant cases (n=29) included 17 slings, 4 abdominal implants, and 8 vaginal implants. Indications for surgical removal were urinary obstruction (13), pain (20), and erosion/exposure (17). Compared to control tissues (n=9), vaginal tissue from mesh removal exhibited strong HA and versican staining adjacent to mesh fibers (figure 1). ECM of explant case specimen showed enrichment of HA. Giant cells containing nuclei that were in contact with mesh appeared negative for HA but ECM between lymphocytes was rich in HA. Occasionally, α-SMA positive cells co-localized with versican in characteristic fibrous capsule surrounding mesh fibers. Associated nerves along mesh fibers and within the fibrous capsule stained strongly for versican.
Conclusions: Vaginal tissue in women with symptomatic mesh complications reflected changes in ECM components versican, HA, and α-SMA and suggests that some capsular cells around mesh fibers differentiate into myofibroblasts. Accumulation of space occupying deposits of versican and HA around nerves offers a possible mechanism of pain and merits further investigation. Our data provides further evidence of the fibrotic and inflammatory processes in mesh complications.
|Poster #BS4|OVERACTIVE VOIDING BEHAVIOR IN SURGICALLY-INDUCED MENOPAUSAL MICE EXPOSED TO LIPOPOLYSACCHRIDE (LPS) IS MODULATED BY DISTINCT GENE NETWORK PATHWAYS
Marian Acevedo Alvarez MD¹, Judy Yeh MD², Lery Alvarez-Lugo MS², Ming Lu MD², Warren G. Hill MD³ and Toby Chai MD²
¹CT; ²New Haven, CT; ³Boston, MA
Presented by: Marian Acevedo Alvarez
INTRODUCTION: Overactive bladder (OAB) and urinary tract infection (UTI) increase in incidence during perimenopause. Both conditions respond to estrogen, suggesting they may share common pathophysiological mechanisms sensitive to estrogen signaling. In this study, we aim to ascertain the effect of estrogen on voiding behavior in response to lipopolysaccharide (LPS), surrogate for bacteria, using mice after ovariectomy (OVX) vs sham.
METHODS: Female C57BL6 mice underwent OVX or Sham (n = 10/group). Micturition behavior was characterized using voiding spot assay (VSA) at pre-surgery, 4 weeks post-surgery (prior to LPS exposure), and after each of the three consecutive days of intravesical inoculation with LPS. Mice were euthanized and bladders harvested for histologic examination. Gene expression was characterized using a separate cohort of OVX and Sham mice (n = 9/cohort), with bladders harvested at baseline (Day 0) and after LPS inoculation on Day 1 and Day 3 of serial LPS. Urothelium was isolated from OVX and Sham mice (n = 3/time point) and RNA was extracted for microarray chip hybridization. Ingenuity Pathway Analysis was used to identify specific genes showing patterns of fold changes in expression (cutoff ≥│2│, p < 0.05) paralleling changes in voiding behavior.
RESULTS: OVX mice had increased voiding frequency throughout 3 days of LPS exposure whereas Sham mice almost normalized voiding behavior by day 3 (Fig 1). Flattened rugae were seen on histological evaluation of bladders from OVX mice but not from Sham mice. Out of 34K transcripts, 6 genes were identified showing patterns of changes in expression, correlating with patterns of voiding behavior. For example, each of the specific genes changed in opposite directions in OVX versus Sham animals on day 1 versus day 3, mimicking opposite changes in voiding frequencies. Yet, these genes also changed in the same direction in OVX and Sham at day 1, mimicking similar voiding frequencies.
CONCLUSIONS: Overactive voiding behavior persisted in OVX mice but resolved in Sham mice with repeated LPS challenge. Changes in expression of six urothelial genes were identified that mimicked voiding behavior, serving as potential targets for new OAB and UTI treatment paradigms.
REPEATABILITY OF MOTOR UNIT NUMBER ESTIMATION OF THE EXTERNAL ANAL SPHINCTER
Chuan Zhang MSE¹,²,³, Alvaro Munoz PhD³, Timothy Boone MD, PhD³ and Yingchun Zhang PhD¹,²,³
¹Guangdong Provincial Work Injury Rehabilitation Hospital, Guangzhou, China; ²Department of Biomedical Engineering, University of Houston, Houston, TX, USA; ³Regenerative Medicine Program, Houston Methodist Research Institute, and Department of Urology, Houston Methodist Hospital, Houston, TX, USA
Presented by: Yingchun Zhang
Objectives: Impairment to the functional innervation of the external anal sphincter (EAS) can be a consequence of impairment to the central or peripheral nervous system and injury to the sphincter musculature. A novel EAS motor unit number estimation (MUNE) technique was developed in our lab to investigate the global EAS innervation in a female rat model, and was further validated by immune-histochemical staining. The aim of this study is to evaluate the test-retest reproducibility of our previously developed approach.
Methods: A total of 7 female Sprague-Dawley rats were tested in this study, including intact (n=2) and sham (n=5, laminectomy at sacral spinal cord but no injury) animals. Sham animals were given post-surgery care and were tested four weeks after surgery. The MUNE approach incorporated trans-vaginal pudendal nerve stimulation, intra-rectal surface EMG recordings, and a statistical MUNE algorithm. MUNE was performed for each animal under anesthesia (urethane, 1.2 g/kg), followed by a 10 min break. Test-retest reproducibility was then evaluated by removal and re-insertion of stimulation and recording probes, and repeating the aforementioned MUNE procedure. Coefficient of variation (CV) was used to quantify the repeatability of compound muscle action potential (CMAP), averaged single motor unit potential (SMUP), and MUNE values determined during offline processing. CMAP was calculated as the averaged peak amplitude across four surface EMG channels.
Results: MUNE and repeatability tests were successful in all 7 animals. An averaged CMAP value of 2029.24±440.56 µV, a mean SMUP size of 55.76±11.62 µV and a mean MUNE of 38±9 was found across all animals. The CV for CMAP, SMUP and MUNE is 4.60±6.14% (1.34% to 19.38%), 7.44±5.57% (from 1.47% to 17.1%) and 7.01±5.02% (from 2.48% to 17.37%).
Conclusions: The results demonstrated marked repeatability of the presented EAS MUNE model, enabling reliable long-term tracking of neuromuscular alterations in the EAS in the presented rodent model. Future explorations will include applying the MUNE approach to a rodent spinal cord injury model to investigate the chronic impact of spinal impairment.
Support: This work was supported by the Brown foundation, the Houston Methodist foundation, NIH R00DK082644, Guangdong Provincial Work Injury Rehabilitation Hospital and the University of Houston.
|Poster #BS6|OPTIMIZING TENSILE STRENGTH USING DIFFERENT COLLAGEN-BASED NANOPARTICLES FOR ELECTROCHEMICAL ALIGNMENT GRAFT FABRICATION OF BIOTEXTILES DESIGNED FOR INCONTINENCE AND PELVIC RECONSTRUCTIVE SURGERY
Raymond Rackley MD¹, Nicole Edwards BME², XingGuo Cheng PhD², Brad Gill BME, MD³ and David Staskin MD⁴
¹Cleveland, OH; ²SouthWest Research Institute; ³Cleveland Clinic; ⁴Steward Health Tufts University of Medicine
Presented by: Raymond Rackley
Introduction and Objectives: We previously reported that biofabricated devices of collagen-based nanoparticles share similar properties to autografts in promoting functional tissue repair and regeneration. We hypothesize that techniques using planar electrochemical alignment (ECA) of 2 different collagen-based nanoparticles for pelvic devices may have inherent biomechanical characteristics that better replicate the biological strength of autografts. Our specific aim of this basic science study was to compare the fracture stress of different concentrations of acid and pepsin soluble collagen-based cross-linked collagen sheets fabricated by the ECA method.
Methods: Graft sheets of biotextile fabrication using solubilized collagen in various concentrations of acid versus pepsin derived treatment was performed by controlled molecular assembly using planar ECA that moves proteins in a pH gradient produced by the electrolysis of water. Fracture stress, the point at which the biotextile sheet failed, was determined.
Results Obtained: See summary graphic below; biofabricated graft sheets using the ECA method had a fracture stress well within the range of values reported for rectus fascia and fascia lata autografts. While the ECA fabricated sheets are thin, they showed excellent tensile strength with a high fracture stress. Very little deformation was seen before complete failure was noted (completely torn). Furthermore, the combination of pepsin and acid solubilized collagen nanoparticles provided the best overall results in considering both the dry lyophilized and post-lyophilized hydrated state.
Conclusions: Novel collagen-based nanoparticles can be easily fabricated from a combination of pepsin and acid derived solubilization via ECA using planar electrodes into planar sheets with excellent biomechanical properties. These findings provide the generation of stress-strain curve studies to further enhance the balance between additional biomechanical, bioactive and biocompatibility features of biofabrics.
Funding: Southwest Research Institute®; Armed Forces Institute of Regenerative Medicine; BioFabrix, LLC
|Poster #BS7|DIFFERENTIAL PROTEIN EXPRESSION IN PATIENTS WITH UCPPS: A MAPP STUDY
Jennifer Anger MD MPH¹, A. Lenore Ackerman MD Ph D², Weston Spivia MS², Irene van den Broek Ph D², Daniel Crear MS³, Karyn Eilber MD², Michael Freeman Ph D², Jayoung Kim Ph D², Qin Fu Ph D² and Jennifer Van Eyk Ph D²
¹Cedars-Sinai Medical Center; ²Cedars-Sinai Medical Center, Los Angeles, California; ³Virginia Institute of Marine Science, Gloucester Point, Virginia
Presented by: Jennifer Anger
Introduction/Objectives: Urologic chronic pelvic pain syndrome (UCPPS) encompasses both interstitial cystitis/bladder pain syndrome and chronic prostatitis/chronic pelvic pain syndrome. A lack of understanding of the molecular mechanisms underlying UCPPS has been a challenge and dilemma for diagnosis and treatment leading to a delay in basic and translational research focused on biomarker and drug discovery, clinical therapy, and preventive strategies. Hoping to identify a specific diagnostic signature of UCPPS, our hypothesis is that UCPPS is associated with specific protein patterns in the blood.
Methods: We collected serum samples from 400 patients who participated in the MAPP network. We applied multiple reaction monitoring mass spectrometry (MRM-MS) methods for 72 pre-selected targeted proteins that are involved in many diseases and inflammatory processes. The largest categories of study were control vs UCPPS. We also matched patients by pain severity, gender, pelvic pain vs. pelvic pain and beyond (widespread pain). These were processed and analyzed.
Results Obtained: Proteins had significant differential expression across five categories, including age, sex, cohort (control vs. UCPPS), and urinary severity. One protein, sex hormone binding globulin, was differentially expressed in Rand Interstitial Cystitis Epidemiology (RICE) subtypes, specifically pain with bladder filling. We also identified interactions between proteins and their overlap across comparison groups (Figure 1). Many markers had overlap between, for example, urinary severity and the presence of UCPPS (vs. control). Some markers were seen across three or more comparisons.
Conclusions: Although validation studies are needed and underway, the targeted analysis of 72 proteins, which are involved in multiple pathways including inflammation, appears to distinguish patients with UCPPS vs. controls. Depending on the peptide it also distinguishes between sex, age, and urinary severity. Understanding the signaling networks perturbed in UCPPS will open new avenues to the identification of novel biomarkers and, equally important, drug targets.
REGULATION OF CONJUGATIVE TRANSFER OF B-LACTAM RESISTANCE FROM UROPATHOGENIC STRAINS OF ESCHERICHIA COLI
Tatyana Sysoeva PhD and Lingchong You PhD
Duke University, Durham, NC
Presented by: Tatyana Sysoeva
Introduction and Objectives: Multidrug resistant uropathogens are becoming increasingly wide-spread making it difficult to treat common diseases such as urinary tract infections (UTI). Antibiotic resistance genes are often associated with mobile plasmids that can transfer directly from cell to cell through conjugation, promoting spread of these plasmids and adding to global burden of antibiotic resistance. We set to investigate how uropathogenic Escherichia coli contribute to the spread of antibiotic resistance genes.
Methods: To mimic the interaction of pathogenic E. coli strains with human commensal strains, we screened a set of multidrug resistant clinical isolates of pathogenic Escherichia coli obtained from patients with UTI, for their ability to donate the β-lactam antibiotic resistance gene to laboratory E. coli strains via conjugation. Mating assays were performed for clinical isolates and for known conjugative plasmids from seven different groups. Donor cultures were grown to the indicated growth stage and concentrated to equal densities before the mating assay. Plasmids were isolated and assayed by agarose gel electrophoresis. Involvement of cAMP signaling in regulation was tested by comparing wild-type donor cells with Δcya background.
Results: Within the tested set of 34 antibiotic resistant uropathogenic isolates we identified 17 that efficiently donated their resistance into non-pathogenic E. coli. Each isolate contained one to five separate plasmids ranging in size from 3 to over 100 kb. Plasmids from different groups showed strong growth-phase dependent regulation of conjugation that was classified into three major classes. The tested conjugating uropathogenic isolates exhibited only one type of regulation with faster transfer from exponential cells. We observed that transfer regulation in the diverse systems hinges on cAMP signaling that assists the transition from exponential to stationary culture.
Conclusions: Clinical isolates of multidrug resistant uropathogenic E. coli contain multiple extrachromosomal elements that transfer resistance genes via conjugation. Donor- and plasmid-derived factors define the efficiency of conjugative transfer and these factors can be targeted to develop strategies to prevent transfer of antibiotic resistance genes between bacteria and halt the global spread of these mobile plasmids.
Funding Sources: K12 DK100024 - Duke KURe Program to TS; NIH, David & Lucile Packard Foundation, and ARO grants to LY.
THE UROPATHOGENIC ESCHERICHIA COLI PILUS USHER CONTROLS PILUS ASSEMBLY THROUGH A 2-STEP VERIFICATION PROCESS DURING ACTIVATION
Glenn Werneburg PhD, Hemil Chauhan BS, Nadine Henderson MS and David Thanassi PhD
Stony Brook University School of Medicine, Stony Brook, NY
Presented by: Glenn Werneburg
Uropathogenic strains of Escherichia coli use the chaperone/usher (CU) pathway to construct adhesive surface structures termed pili or fimbriae. Pili facilitate binding to bladder and kidney epithelial cells, thereby promoting urinary tract colonization and infection. The CU pilus biogenesis pathway requires a periplasmic chaperone protein and an outer membrane usher protein. The usher catalyzes the exchange of chaperone-subunit for subunit-subunit interactions, and promotes ordered polymerization and secretion of subunits into the mature pilus fiber. An essential and well-regulated step of pilus assembly is the activation of the usher, in which the usher undergoes a transformation from a plug-gated membrane pore to an engaged assembly machine.
To gain a mechanistic understanding of how the usher is activated and controls the assembly of pili, we monitored pilus subunit interactions with different usher domains using genetic, biochemical, and biophysical approaches including Förster Resonance Energy Transfer (FRET).
Using an inter- and intramolecular FRET-based affinity approach, we identified the necessary conditions for usher activation. We identified particular regions of the activating pilus subunit (adhesin) that are essential for discrete steps of the activation process. Specifically, we demonstrated that the C-terminal domain (pilin domain) of the activating subunit is necessary for subunit recruitment to the usher platform (Kd=194 nM), and that the N-terminal domain (lectin domain) of the activating subunit is necessary for subsequent usher plug expulsion (act1/2=4 nM) and priming for pilus assembly (Kd=4 nM). Further, using a mutagenesis approach, we were able to demonstrate that the addition of the lectin domain is sufficient to confer usher-activating potential to a non-activating pilus subunit.
Our results suggest that the usher employs a “2-step verification” process in which it recognizes and verifies both the N and C domains of the initiating adhesin subunit, each during a discrete step in the activation process. This process may ensure that every assembled pilus has an adhesin at its tip, in a location to facilitate urinary tract adhesion and UTI. These discoveries open avenues for informed drug design to target these newly-elucidated mechanistic steps, thus potentially preventing or treating UTI. Our FRET-based approaches are poised for the screening of molecules to disrupt this process.
THE ROLE OF PDGFRα+ CELLS IN CYCLOPHOSPHAMIDE−INDUCED DETRUSOR OVERACTIVITY
Haeyeong Lee PhD¹, Byoung Koh BS², Lauren Peri BS², Kenton Sanders PhD² and Sang Koh MD,PhD²
¹University of Nevada, Reno, School of Medicine, Department of Physiology and Cell Biology; ²University of Nevada, Reno, School of Medicine, Department of Physiology and Cell Biology, Reno, NV
Presented by: Haeyeong Lee
INTRODUCTION: Cyclophosphamide (CYP) is known to cause cystitis in humans, and CYP-induced cystitis in rodents has been used to investigate this disorder because there are many features in common with cystitis occurring in patients treated with CYP. The changes in CYP-injected bladders produce functional changes that are thought to occur via urothelial and neural effects. However, functional changes in detrusor muscles due to CYP treatment have not been characterized. In previous reports, we demonstrated the role of detrusor PDGFRα+ cells during bladder filling. In the present study, we examined the molecular and functional changes occurring in detrusor PDGFRα+ cells in CYP-induced detrusor overactivity.
METHODS: CYP was injected intraperitoneally for 4 injections in a 7days in PDGFRα+/eGFP and SMC/eGFP mice. We harvested the detrusor muscle on day 8 and dispersed the cells for the fluorescence activated cell sorting. Sorted PDGFRα+ cells and smooth muscle cells (SMCs) were used for molecular study to compare the changes in transcripts between CYP−injected and control group. Immunohistochemistry, mechanical contractility, patch clamp and ex vivo cystometry were also performed.
RESULTS: qPCR revealed that CYP−injected detrusor muscle decreased Pdgfra expression with an increase in Il6 and Tnfα. Transcriptional changes in sorted PDGFRα+ cells from CYP−injected PDGFRα+/eGFP mice showed Pdgfα and Kcnn3 (SK3) transcripts were decreased compared with saline−injected control. Sorted SMCs from SMC/eGFP mice did not show detectable changes. Immunohistochemistry showed that SK3 and PDGFRα immunoreactivity were downregulated in CYP−injected detrusor muscle. In mechanical experiments, apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP−injected detrusor muscles. In ex vivo cystometry, increased spontaneous transient contractions and less apamin sensitivity were observed in CYP−injected bladder. CyPPA (a SK activator) induced hyperpolarization and activated SK currents in detrusor PDGFRα+ cells in control PDGFRα+ cells. SK current density and hyperpolarization responses to CyPPA was greatly reduced in CYP-injected PDGFRα+ cells
CONCLUSIONS: In conclusion, we found that CYP−induced detrusor overactivity is resulted from loss or downregulation of PDGFRα and SK channels in detrusor PDGFRα+ cells. These results provide novel mechanisms of functional role of PDGFRα+ cells on detrusor overactivity. (Supported by NIH RO1 DK098388)
|Poster #BS11|COMPARISON OF IRON-DEPENDENT REGULATION OF SURFACE MOTILITY IN UROPATHOGENIC AND NONPATHOGENIC ESCHERICHIA COLI
Parker McDill BS, MS¹, Larry Reitzer BS, PhD¹ and Philippe Zimmern MD²
¹UTD; ²UT Southwestern Medical Center
Presented by: Parker McDill
Introduction and Objectives: Iron is an essential nutrient for growth as well as a major regulator of virulence factor synthesis. Swarming is a rapid form of bacterial surface motility and likely plays a role in the development of urinary tract infections. In a nonpathogenic strain of Escherichia coli, we have observed iron-dependent regulation of swarming. Our goal was to determine whether iron also regulates swarming for clinical isolates of uropathogenic Escherichia coli (UPEC).
Methods: Strains used were the nonpathogenic W3110 and the UPEC strains UTI 89 (acute cystitis), RUTI 12 (chronic cystitis), and CFT073 (pyelonephritis). To assay swarming, 1μL of an overnight culture grown in liquid swarm media (1% tryptone, 0.25% NaCl, 0.5% glucose) was inoculated in the center of a plate with 25mL solidified swarm media (0.45% Eiken agar) and placed in a 37°C incubator for 36 hours. Low iron media was prepared by adding 2,2’-bipyridal, an iron chelator, to a concentration of 100μM in freshly autoclaved media. Swarming was measured by the distance (mm) of outward movement from the center.
Results obtained: When iron was not the limiting nutrient, all four strains swarmed. Whereas both UTI 89 (90mm) and RUTI 12 (90mm) swarmed to the edge of iron-sufficient swarm plates, CFT073 (39mm) and W3110 (47mm) did not. However, under iron-limiting conditions, only W3110 (30mm) and UTI 89 (90mm) were capable of swarming. In contrast, neither RUTI 12 (6mm) nor CFT073 (9mm) displayed significant movement.
Conclusions: The striking differences observed in these swarm phenotypes clearly indicate strain-dependent differences in the regulation of swarming motility. Such differences are consistent with the hypothesis that strain-specific adaptations alter regulatory networks. For iron in particular, changes in the network of iron-responsive genes would affect not only swarming motility but also virulence. Therefore, swarming proficiency in iron-limited environments could favor the initial ascension of the urethra (UTI 89), and the subsequent loss of this ability may be concomitant with the establishment of long-term infections, such as chronic cystitis (RUTI 12) and pyelonephritis (CFT073).
Financial Funding: none
|Poster #BS12|A RELIABLE, SENSITIVE AND FAST ENZYMATIC METHOD TO MEASURE D-MANNOSURIA IN WOMEN.
Iti Mehta BS, MS¹, Larry Reitzer BS, PhD¹ and Philippe Zimmern MD²
¹UTD; ²UT Southwestern Medical Center
Presented by: Iti Mehta
Introduction and Objective: Many women with recurrent urinary tract infections (RUTIs) take over-the-counter D-mannose supplements. The rationale is that D-mannose has been shown to block bacterial fimbriae adhesion to the bladder wall mucosa in an animal model, thereby possibly reducing the frequency and severity of RUTIs. Studies have measured the anti-adhesion activity in urine through serial dilution after ingestion of oral D-mannose, but there is no published technique to specifically and sensitively measure D-mannose in urine. The objective was to develop a precise, sensitive, and fast enzymatic method to measure D-mannosuria in women.
Methods: The reaction was performed in three stages. In the first stage, a small urine sample (0.1 ml) was added to the reaction assay mixture that contained hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P DH), Mg++, ATP, and NADP+. Glucose is stoichiometrically converted to glucose-6-phosphate (G6P) and 6-phosphogluconate (6-PG) with the formation of NADPH, which is measured with a spectrophotometer at 340 nanometer. In the second stage, G6P isomerase (Pgi) was added, which converts fructose-6-phosphate to G6P, and subsequent change in A340 measured fructose. In the third stage, ManA was added, and the subsequent increase in A340 measured mannose. Five nanomoles sugars (delta A340 of 0.03) were reliably detected by this method. The whole assay took about 30 minutes.
Results Obtained: A known concentration of D-mannose was added to human urine to confirm the accuracy of the method and show that urine does not inhibit the reactions. Two grams D-mannose was given orally to two healthy volunteers. Baseline and hourly samples after D-mannose 2 gm oral intake were then obtained. To allow sample comparison, all urinary concentrations of D Mannose were reported as a ratio to the urinary creatinine level (Figure 1).
Conclusions: A reliable method of measuring D-mannosuria was devised which may prove useful to determine the efficacy and optimize the intake (dose, intake frequency, elimination ratio) of D-mannose in women suffering from RUTIs.
MYOGENIC MECHANISMS OF DETRUSOR OVERACTIVITY IN SPINAL CORD INJURY
Haeyeong Lee PhD¹, Byoung Koh BS², Robert Corrigan BS², Andrew Yanez ², Tong Zhou PhD², Kenton Sanders PhD² and Sang Koh MD,PhD²
¹University of Nevada, Reno, School of Medicine, Department of Physiology and Cell Biology; ²University of Nevada, Reno, School of Medicine, Department of Physiology and Cell Biology, Reno, NV
Presented by: Haeyeong Lee
INTRODUCTION: The bladder has the unique capability of maintaining a low muscle excitability during filling. We discovered and characterized an entirely novel control mechanism that regulates detrusor excitability. PDGFRα+ cells supply a powerful inhibitory mechanism to the bladder during filling. This mechanism is composed of the following molecular and functional components: 1) PDGFRα+ cells display strong expression of SK3 channels (Kcnn3). 2) Hyperpolarization due to activation of SK channels in PDGFRα+ cells exerts a membrane potential-stabilizing effect on detrusor muscles. 3) Expression of TRPV4 channels provides a stretch-dependent source for Ca2+ entry and activation of SK channels. 4) Genetic deactivation of TRPV4 and SK3 channels result in bladder overactivity. The hypothesis of this present study is the loss or defects in PDGFRα+ cells or in key molecular components of the inhibitory regulation provided by PDGFRα+ cells in spinal cord injury (SCI) leads to detrusor dysfunction and development of an overactive phenotype.
METHODS: SCI was induced by complete compression of T13-L1 spinal cord. Experiments were performed on 24 hr, 48 hr and 72 hr after surgery. We employed molecular approaches (RNAseq, qPCR and protein studies) and ex vivo cystometry.
RESULTS: In ex vivo cystometry, SCI bladder revealed an increase in the amplitude and frequency of transient contractions (TCs; relevant to non-voiding contractions in in-vivo cystometry) during filling. TCs increased in SCI bladders. Effects of a SK blocker (apamin) and a SK channel activator (SKA31) on TCs were reduced in SCI mice compared to control suggesting downregulation of SK channels in SCI bladder. qPCR, immunohistochemistry and immunoblot showed the loss of PDGFRa and downregulation of SK channels. RNAseq and qPCR data showed the apoptosis-related geneset score was significantly increased in SCI detrusor muscles.
CONCLUSIONS: These findings support that loss or downregulation of PDGFRα+ cells and SK channels in SCI detrusors might involve the development of detrusor overactivity from SCI. (Supported by NIDDK, RO1 DK098388)
|Poster #BS15|CHARACTERIZATION OF RELAXIN RECEPTOR EXPRESSION IN HUMAN BLADDER SMOOTH MUSCLE CELLS AND EVALUATION OF ITS EFFECT ON TISSUE REMODELING AND FIBROSIS
Edward Diaz MD, Mason Briggs BS, Yan Wen MD, Amy Dobberfuhl MD and Bertha Chen MD
Stanford University School of Medicine
Presented by: Edward Diaz
Introduction: Relaxin has the ability to inhibit fibrotic pathways and modulate the extracellular matrix and is being studied as a therapy for fibrotic medical conditions such as scleroderma, and pulmonary fibrosis. Currently there is no published data on relaxin’s role in the human bladder.
Objective: Characterize relaxin receptor expression in human bladder smooth muscle cells (SMCs) and assess its effect on tissue remodeling and fibrosis.
Methods: Primary human bladder SMCs were obtained commercially. We assessed relaxin/insulin like family peptide receptor 1 (RXFP1) and RXFP2 expression using quantitative reverse transcriptase-PCR (qRT-PCR), and immunohistochemistry (IHC).
Human bladder SMCs were grown in media for less than 7 passages. Cells were starved and administered various concentrations of relaxin 2 (0, 1, 10, 100 ng/mL). Cell Lysate and supernatant were collected at 24 and 48 hours. RNA was obtained from cell lysate and used for qRT-PCR. Protein expression from cell lysate and supernatant were used for western blot and zymogram assays. Assessed proteins included: elastin, collagen 1, collagen 3, Transforming growth factor-beta 1 (TGF-beta 1), matrix metalloproteinases (MMP) 2, MMP 9, tissue inhibitors of metalloproteinases (TIMP) 1, and TIMP 3.
Results: On immunohistochemistry, primary human bladder SMCs stained positive for RXFP1. This was confirmed with qRT-PCR. We did not detect significant expression of RXFP2 on qRT-PCR or immunohistochemistry. qRT-PCR on RNA obtained from 24 hour cell lysate revealed no significant change in expression for collagen 1, collagen 3, TGF-beta 1, MMP 9, TIMP 1, and TIMP 3. There was a trend towards increased expression of MMP-2 and elastin. MMP-2 Zymogram for cell lysate and supernatant at 24 hours did not display obvious pattern of increased expression, however, 48 hours illustrated statistically significant increased expression of latent and active forms of MMP-2. See Figure.
Conclusion: Human bladder SMCs express RXFP1, the receptor for relaxin 2. Our data demonstrates relaxin can have an effect on bladder SMCs at concentrations at 1 ng/mL in vitro. MMP-2 has a dose response to relaxin that is most evident at 48 hours.
Source of Funding: None
NERVE STIMULATION INCREASES VOIDING EFFICIENCY IN A NOVEL MODEL OF DETRUSOR UNDERACTIVITY
Eric Gonzalez PhD and Warren Grill PhD
Department of Biomedical Engineering, Duke University, Durham, NC
Presented by: Eric Gonzalez
Detrusor underactivity (DUA) is an understudied health concern that affects up to 45% of men and women. The clinical management of DUA is inadequate and fails to improve the quality of life of these patients. The limited availability of animal models that exhibit the integrated pathophysiology of DUA impedes the development of new therapeutic approaches. The current studies characterized the bladder function of an obesity model of DUA and investigated neuromodulation as a management option to restore efficient bladder emptying. Eight-week old female obese-prone (OP) and obese-resistant (OR) rats were purchased from Charles River (Boston, MA). OP and OR rats were fed a 45% fat diet from 9-21 weeks and a 60% fat diet from 21-24 weeks. Serum was collected from the tail vein for metabolic analysis at 8 and 24 weeks. Chronic bladder function (voided volume and voiding frequency) of OP rats was assessed twice per week from 8-24 weeks in a metabolism cage. At 24 weeks, OP and OR rats were anesthetized with urethane (1.2 g/kg s.c., supplemented as needed) and underwent acute single-trial cystometry. A paddle with platinum iridium contacts was placed on the EUS to record EMG signals and a bipolar microcuff was placed around the motor or sensory branch of the pudendal nerve or the pelvic nerve for electrical stimulation. Following diet-induced obesity (DIO), OP rats weighed more than OR rats and had normal blood glucose but developed hyperinsulinemia and hypertriglyceridemia. During the chronic monitoring of bladder function, the voiding frequency and voided volumes of OP rats remained relatively constant when normalized to water intake. OP rats, however, exhibited DUA and urinary retention following DIO in acute cystometry. Compared to OR rats, OP rats had increased volume threshold, decreased peak micturition pressure (p ≤ 0.001), decreased voiding efficiency (p ≤ 0.0001), and decreased EUS EMG activity during voiding. Patterned electrical stimulation of the motor branch of the pudendal nerve increased voiding efficiency two-fold in OP rats (p ≤ 0.05), whereas, stimulation of the sensory branch of the pudendal nerve did not alter voiding efficiency. OP rats also had no change in voiding efficiency and decreased evoked contraction amplitude with electrical stimulation of the pelvic nerve. This animal model may be used to understand the pathophysiology of DUA and establish the efficacy of neuromodulation to recover efficient bladder emptying with urinary retention.
AMPLITUDE EFFECTS OF SACRAL NEUROMODULATION IN THE FULLY CONSCIOUS OVINE MODEL
Thaddeus Brink PhD, Tina Billstrom , Melissa Mattson and Lance Zirpel PhD
Medtronic Inc., Minneapolis, MN
Presented by: Thaddeus Brink
Introduction & Objectives: Sacral neuromodulation (SNM) is a clinically approved therapy for refractory overactive bladder. A fully conscious sheep model was developed to test SNM parameters and novel concepts using changes in bladder capacity. To more fully understand similarities and limitations of the sheep model to clinical usage, we examined the effect of different amplitudes of SNM on bladder capacity.
Methods: Normal, female, Polypay sheep (n=4) were anesthetized and surgically implanted with bilateral InterStim II devices (Model 3058) and leads (Model 3889) in the sacral foramen (S3 or S4). Following recovery, urodynamics were performed weekly. After 5 baseline trials (no SNM), amplitudes of 0.5X, 1X, 2X, 3X motor threshold (MT) or maximum tolerable amplitude (MTA) were applied for 5 trials. MT was defined as the first visual motor reflex and MTA was defined as the maximum amplitude achieved before the sheep showed signs of distress. Amplitudes were presented randomly each week with three weeks obtained at each amplitude. Group data are expressed as average±SEM. Means were compared via 2-way ANOVA with Holm-Sidak pairwise comparisons and p<0.05 considered significant.
Results Obtained: A 2-way ANOVA revealed that only MTA resulted in a significant increase in bladder capacity (df=4, F=5.98, p<0.001). Average bladder capacity during baseline trials was 120±12ml and increased to 200±14ml during SNM. For 0.5MT (137±12ml to 129±9ml), 1XMT (124±11ml to 125±13ml), 2XMT (134±8ml to 128±9ml) and 3XMT (133±9ml to 142±7ml), there was no significant effect of SNM on bladder capacity.
Conclusions: These data show that supra-motor threshold amplitudes of SNM are needed to increase bladder capacity in normal sheep. While the normal ovine model is sufficient for investigating fundamentals of SNM, there may be opportunity to investigate subtler and more closely clinically translatable parameters using an ovine model of bladder overactivity. Future studies may examine SNM effects in different bladder hyperactivity models within the fully conscious sheep.
Funding: This study was funded by and all authors are employees of Medtronic, Inc.
7:00 a.m.-6:30 p.m.
Registration/Information Desk Open
Location: Austin Grand Ballroom Foyer, 6th Floor
7:00 a.m.-5:30 p.m.
Speaker Ready Room Hours
Location: Room 602, 6th Floor
7:30 a.m.-8:30 a.m.
Location: Austin Grand Ballroom Foyer, 6th Floor
12:15 p.m.-1:15 p.m.
Location: Austin Grand Ballroom Foyer, 6th Floor
4:00 p.m.-6:30 p.m.
Fellowship Program Directors Meeting
Location: Room 415 AB, 4th Floor
7:00 p.m.-8:30 p.m.
Welcome Reception with Industry Partners
Location: Austin Grand Ballroom, Salon F-G, 6th Floor
8:30 a.m. - 8:45 a.m.
8:45 a.m. - 10:45 a.m.
Top 10 Basic Science Abstract Presentations
John P. Lavelle, MD, FRCSI
John Malysz, PhD
|8:45 a.m. #1|
EXPRESSION AND FUNCTION OF HETEROMERIC KV7.4/KV7.5 CHANNELS IN HUMAN DETRUSOR SMOOTH MUSCLE
Aaron Provence BSc¹, John Malysz ², Damiano Angoli MSc², Eric Rovner MD³ and Georgi Petkov PhD²
¹Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC; ²Department of Pharmaceutical Sciences, College of Pharmacy, The University of Tennessee Health Science Center, Memphis, TN; ³Department of Urology, Medical University of South Carolina, Charleston, SC
Presented by: John Malysz
Introduction: Detrusor smooth muscle (DSM) facilitates urinary bladder function. Voltage-gated potassium channels (Kv) determine DSM excitability and contractility. Among all Kv channels, Kv7 subtypes have emerged as key regulators of DSM excitation-contraction coupling in guinea pigs, pigs, and rats. Recent studies from our laboratory suggest a role of heteromeric Kv7.4/Kv7.5 channels in guinea pig DSM function, but they are yet to be explored in human DSM.
Methods: DSM specimens were obtained from patients lacking symptoms of overactive bladder (OAB) undergoing open bladder surgeries. DSM whole tissue muscle strips (urothelium-free) and freshly-isolated single DSM cells were prepared and studied by RT-PCR, immunohistochemistry, immunocytochemistry, Western blot, Proximity Ligation Assay (PLA), DSM tissue contractility, and DSM whole-cell perforated patch-clamp electrophysiology.
Results: We found out that human DSM whole tissue and single smooth muscle cells expressed Kv7.4 and Kv7.5 mRNAs and proteins. In situ PLA demonstrated a high degree of spatial co-localization of Kv7.4 and Kv7.5 proteins in single DSM cells. Heteromeric Kv7.4/Kv7.5 complexes were highly expressed in the plasma membrane of DSM cells. In contrast, neither Kv7.4 nor Kv7.5 co-localized with inositol trisphosphate receptors (used as negative controls). Retigabine (Kv7.2-Kv7.5 channel activator) at 10 µM or ML213 (Kv7.4/Kv7.5 channel activator) at 10 µM reduced spontaneous phasic contractions of DSM tissue strips while XE991 (Kv7.1-Kv7.5 channel inhibitor) at 10 µM enhanced DSM contractility. Patch-clamp recordings in DSM cells revealed hyperpolarization induced by retigabine (10 µM) or ML213 (10 µM) and depolarization by XE991 (10 µM). Here for the first time, we recorded native human DSM Kv7 currents (using a ramp protocol) that were activated by retigabine (10 µM).
Conclusion: The close proximity of protein detections for both Kv7.4 and Kv7.5 subtypes in human DSM cells supports a role of native Kv7 channels composed of heteromeric Kv7.4/Kv7.5 channels whose activation leads to DSM hyperpolarization and attenuation of DSM contractility facilitating urine storage. Targeting heteromeric Kv7.4/Kv7.5 channels, displaying properties distinct from homomeric Kv7 channels, provides a potential novel therapeutic approach for the treatment of urinary bladder dysfunction including OAB and underactive bladder.
Funding: NIH R01DK106964 grant to GVP and F31DK104528 fellowship to AP.
|8:57 a.m. #2|OVEREXPRESSION OF ESTROGEN RECEPTOR β IN UROTHELIUM PROTECTS AGAINST UROPATHOGENIC E. COLI URINARY TRACT INFECTION
Judy Yeh MD¹, Marian Acevedo MD¹, Lery Alvarez MS¹, Ming Lu MD¹, Warren Hill PhD² and Toby Chai MD¹
¹Yale, New Haven, CT; ²Beth Israel Deaconess, Boston, MA
Presented by: Toby Chai
Introduction: Host factors in female UTI defense include estrogen. A prior study showed that estrogen’s UTI protective effect on bladder urothelial cells grown in culture (in vitro experiments) was through ERβ and not ERα. We hypothesize that overexpression of urothelial ERβ in a transgenic mouse would show protection against UTI development (in vivo model).
A transgene containing a UPII promoter (urothelial restriction) linked to the ERβ gene was inserted into the C57BL6 mouse (WT) genome to create a urothelially restricted ERβ overexpressing mouse (uERβ-OE+). Female mice, 12 weeks old, were used derived from 3 cohort populations – WT, uERβ-OEneg (non-transgene carrying littermates) and uERβ-OE+. UTI was created by transurethral inoculation of uropathogenic E. coli (UPEC) at a dose of 1 x 10e8 colony forming units (CFU) in 50 µL under anesthesia using established protocol. Urine specimens were collected 1, 2, 3, and 4 days post UPEC inoculation and sampled on agar plates in serial dilution to quantitate bacterial load. After the last urine collection on Day 4, mice were euthanized, and their bladders and kidneys were harvested, homogenized, and plated for bacterial counts. Voiding spot assay (VSA) was performed to compare voiding behavior after UPEC inoculation.
qPCR showed that ERβ mRNA was significantly elevated in the uERβ-OE+ urothelium. Bladder histology revealed no differences in the 3 cohorts. uERβ-OE+ mice (n=12) cleared urinary UPEC loads significantly more quickly than the 2 control cohorts (n=24) (Figure A, B). Transgenics also had significantly lower bacterial counts measured in bladders and kidneys (Figure C, D) on Day 4. Control animals voided significantly more on Day 1 after UPEC compared to prior to UPEC inoculation. uERβ-OE+ animals had no change in voiding behavior 1 day after UPEC inoculation.
This is the first demonstration in vivo that increased ERβ signaling on urothelial cells is protective against both lower and upper urinary tract infections. This uERβ-OE+ mouse model will serve as a valuable tool to identify novel pathways that might be harnessed in UTI treatments which leverage host defense responses activated by ERβ.
|9:09 a.m. #3|TRANSGENIC FEMALE MICE WITH ORNITHINE DECARBOXYLASE (ODC) OVER-EXPRESSION RESTRICTED TO UROTHELIUM EXHIBIT OAB VOIDING BEHAVIOR AND INCREASED URINARY CYTOKINES: A TRANSLATIONAL MURINE MODEL OF OAB
Judy Yeh MD¹, Lery Alvarez-Lugo MS¹, Ming Lu MD¹, Warren Hill PhD² and Toby Chai MD¹
¹Yale, New Haven, CT; ²Beth Israel Deaconess, Boston, MA
Presented by: Toby Chai
Introduction: Prior OAB animal models (e.g. cyclophosphamide, acetic acid, bladder obstruction, cerebral infarct, etc.) have lacked translational relevance to idiopathic female OAB. Because human urothelium from female subjects with idiopathic OAB was shown to have increased ornithine decarboxylase (ODC), we hypothesized that urothelial overexpression of ODC would create a novel and translationally relevant OAB animal model.
Methods: A transgene containing a UPII promoter (urothelial restriction) linked to the ODC gene. was inserted into the C57BL6 mouse (WT) genome to create a urothelially restricted ODC overexpressing mouse (ODC+). Three cohorts of 12 week old female mice (ODC+, ODC- [non-transgene carrying siblings in litter] and WT) were used. qPCR for ODC from the urothelium and other tissues was performed. For voiding behavior study, 36 mice (12 in each cohort) were used. Voiding behavior was measured over 4 hours in the dark cycle and void spot assays (VSA) were performed per published method. Urine specimens were collected in animals (n=4 for each cohort) and analyzed with 32-plex murine cytokine ELISA.
Results: Urothelial qPCR showed 20x higher ODC mRNA expression in ODC+ compared to controls. H&E staining of bladder revealed unique intracellular inclusion bodies in umbrella cells in ODC+ animals. ODC+ animals voided significantly more frequently in each of 3 separate days of voiding behavior measurements (Fig. 1A, 1D) with significantly more spots in center of filter papers (Fig. 1B, 1E). The voided volume was not different between the 3 cohorts of animals (Fig. 1C, 1F). Urinary cytokine analyses revealed 6 of the 32 cytokines had a significant elevation in the ODC+ animals: G-CSF, IL-1α, IL-1β, KC (CXCL1), LIX (CXCL5) and VEGF (Fig. 2).
Conclusions: We created a translationally relevant OAB animal model. This ODC+ animal had OAB phenotype including increased voiding frequency and increased urinary cytokine expression of six cytokines. The ODC+ animal is a valuable and novel tool to study urothelial dysregulation contribution to OAB bladder behaviour phenotype and represents a beside to bench paradigm for studying OAB pathophysiology.
|9:21 a.m. #4|FLOW STUDIES IN THE ISOLATED PERFUSED WORKING PIG BLADDER DEMONSTRATE PRESERVATION OF TISSUE OXYGENATION DESPITE DECREASING VASCULAR FLOW: POTENTIAL MECHANISMS OF UNDERACTIVE BLADDER
Uzoma Anele MD¹, Andrew Tracey MD¹, Andrew Colhoun MD¹, John Speich PhD², Paul Ratz PhD³ and Adam Klausner MD¹
¹Virginia Commonwealth University Medical Center, Richmond, VA; ²Virginia Commonwealth University, Richmond, VA; ³Virginia Commonwealth University School of Medicine, Richmond, VA
Presented by: Uzoma Anele
Introduction and Objectives.Detrusor dysfunction, particularly underactivity is an increasingly recognized phenomenon; however, contributing underlying mechanisms are poorly understood partly due to the lack of appropriate functional models for adequate study. The role of vascular resistance in maintaining tissue oxygenation (TpO2) upon reductions in tissue perfusion in bladder remains to be determined. Therefore, we developed a model to investigate the dependencies of vascular perfusion pressure and TpO2 on perfusion flow in isolated pig bladder.
Methods.Bladders from local abattoirs were harvested and prepared. Vesical arteries were cannulated and perfused with a Krebs buffer. Intravesical pressure was measured via a cannulated ureter. After 1hr equilibration at perfusion flow of 20ml/min, the bladder was filled to 500mL via urethral catheter and re-equilibrated for 30min. Vascular resistance was assessed over a range of vascular flows by a stepwise decrease in flow rate by 5ml/min increments to 0ml/min and observed for 15-30min following each perturbation. Intravesical pressure, perfusion pressure, and TpO2 were recorded.
Results.Bladders from 11 pigs were studied. Perfusion pressure decreased linearly with decreasing flow rate (Fig1a n=11, p<0.01). Intravesical pressure also decreased with flow rate (Fig1b n=11, p<0.01). However, TpO2 remained stable (Fig1c n=7, p=0.24). The lack of change in vascular resistance was not due to absence of vascular reactivity in this model because perfusion pressure decreased or increased with intravascular nitroglycerin or phenylephrine (Fig1d n=2).
Conclusions.This suggests that autoregulation does not occur in bladder vasculature. However, maintenance of bladder TpO2 despite decreasing flow indicates involvement of an active, compensatory mechanism. Although responsivity to vasoactive agents suggests capacity for vascular tone regulation, vascular resistance was constant over flow ranges of 20-0ml/min, suggesting that isolated pig bladder maintains TpO2 by increased capillarity rather than vascular autoregulation. Further study is necessary to define these regulatory mechanisms which may help identify novel pathways involved in understanding or treating underactive bladder.
|9:33 a.m. #5|
UNDERSTANDING THE PATHOGENS RESPONSIBLE FOR RECURRENT URINARY TRACT INFECTIONS IN POSTMENOPAUSAL WOMEN
Nicole De Nisco BS, PhD¹, Luming Chen BS², Marcela de Souza Santos PhD², Kelli Palmer PhD³, Kim Orth BS, MS, PhD² and Philippe Zimmern MD²
¹UT Southwestern Medical Center and Howard Hughes Medical Institute; ²UT Southwestern Medical Center; ³UTD
Presented by: Nicole De Nisco
Introduction and Objectives: Work with murine models has shown that uropathogenic Escherichia coli (UPEC) is able invade the bladder urothelium and form intracellular reservoirs, which are hypothesized to cause RUTI in humans (1). However, this hypothesis has not been tested in RUTI patients. To that intent, we analyzed a cohort of postmenopausal women undergoing cystoscopy with fulguration of trigonitis (CFT) for treatment of RUTI after multiple failed antibiotic courses (2).
Methods: Following IRB approval, bladder biopsies and urine were obtained from consenting women meeting study criteria for antibiotic-refractory RUTIs and with office-based evidence of chronic cystitis on flexible cystoscopy. All samples were obtained in the operating room under anesthesia following a rigorous protocol. Antibiotics were discontinued for at least one week prior to CFT. Tissue and urine samples were analyzed using both culture-based and molecular approaches to determine if intracellular bacterial reservoirs are present within the bladder urothelium. Our methods included aseptic microbial culture, 16S rRNA fluorescence in situ hybridization, 16S rRNA variable (v4) region Illumina deep sequencing, as well as basic molecular biology and histological techniques.
Results Obtained: Our preliminary studies of six post-menopausal RUTI patients with extensive cystitis cystica or pancystitis indicated that a variety of Gram-negative and Gram-positive bacteria were present inside the bladder urothelium. Strikingly, negative urine cultures at the time of CFT were noted in some patients despite positive bacterial tissue sample findings.
Conclusions: So far, we have found that diverse and unexpected bacterial species can invade the urothelium of severely infected post-menopausal RUTI patients. These intracellular bacterial reservoirs may explain the observed cycles of recurrence.
References: 1. Proc Natl Acad Sci U S A 101, 1333-1338 (2004). 2. Urol Sci 25, 1-8 (2014).
Financial Funding: This work was funded by the National Institutes of Health (NIH) grant R01-AI056404, the Welch Foundation grant I-1561, and Once Upon a Time
|9:45 a.m. #6|
STRESS-INDUCED BLADDER HYPERSENSITIVITY, HINDPAW ALLODYNIA, AND DEPRESSION-LIKE BEHAVIOR IN AN ANXIETY-PRONE STRAIN OF MICE
Pau Yen Wu ¹, Xiaofang Yang MD¹, Douglas Wright PhD¹ and Julie Christianson PhD²
¹University of Kansas Medical Center, Kansas City, KS; ²University of Kansas Medical Center
Presented by: Julie Christianson
Introduction and objectives: Patients with chronic pelvic pain disorders commonly suffer from comorbid mood disorders, such as anxiety, panic disorder, and depression. This comorbidity has been associated with altered limbic regulation of the hypothalamic-pituitary-adrenal (HPA) axis, which initiates the stress response and influences the perception of pain. Exposure to stress can initiate and/or exacerbate symptomology in these patients. To explore the mechanisms underlying stress-induced comorbidity between chronic pain and mood disorders, we are testing the hypothesis that chronic stress exposure can increase urogenital sensitivity, hindpaw allodynia, and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety.
Methods: Female, 12-week-old A/J mice were exposed to repeated foot shock stress, or sham exposure, for 10 continuous days and tested 1 or 28 days later for visceromotor response (VMR) during urinary bladder distension (UBD), hindpaw mechanical sensitivity, sucrose preference, nest building behavior, mast cell degranulation, and serum corticosterone (CORT) levels.
Results obtained: Mice that underwent foot shock stress displayed a significant increase in VMR during UBD, compared to sham mice, only at the 1 day time point. At both time points, the foot shock group had significantly decreased mechanical withdrawal thresholds in the hind paw compared to their baseline and sham group measurements. Sucrose preference and nest construction tests revealed that anhedonic behavior was displayed immediately after foot shock exposure. Both sham and foot shock groups had histological evidence of very high rates (>80%) of mast cell degranulation. Finally, foot shock exposure induced a significant increase in serum CORT levels.
Conclusions: Ten days of foot shock stress induced acute bladder hypersensitivity, immediate and long-lasting hindpaw allodynia, anhedonic behavior, and increased serum CORT levels. Together, these data suggest that foot shock stress in A/J mice may provide a useful tool for understanding the connections between stress, mood disorder, and chronic pelvic pain. Future studies will investigate long-term changes in anhedonic behavior, gene expression within the limbic structures known to regulate the HPA axis, and potential interventional and pharmacological therapies in this novel preclinical model.
Supported by NIH grants DK099611, DK103872, and NS043314.
|9:57 a.m. #7|
SUSTAINED INHIBITION OF BLADDER FUNCTION IS EVOKED BY SAPHENOUS NERVE STIMULATION: AN EVALUATION OF A CONTINUOUS URODYNAMIC MODEL IN ANESTHETIZED RATS
Zainab Moazzam BS¹ and Paul Yoo PhD²
¹Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada; ²University of Toronto
Presented by: Paul Yoo
OBJECTIVES: As a potential new treatment for overactive bladder, we recently described a novel bladder-inhibitory reflex evoked by saphenous nerve stimulation in anesthetized rats. In this study, we sought to characterize the inhibitory effects of longer-duration SAFN stimulation using a continuous urodynamic model.
METHODS: Non-survival experiments were conducted in 11 urethane-anesthetized rats. Following a midline incision, the bladder was catheterized and connected to a pressure transducer connected in series with an infusion pump. The bladder was continuously infused with saline (0.08 ml/min) until stable rhythmic bladder contractions were achieved. The SAFN was instrumented with a bipolar nerve cuff electrode. The stimulation amplitude was set at 25 uA, and 40-minute stimulation trials were applied at frequencies of 10 Hz and 20 Hz. The measured basal pressure (BP), inter-contraction interval (ICI) and contraction amplitude (CA) were analyzed during both the intra-stimulation and post-stimulation periods. Each variable was normalized to the baseline of each experiment.
RESULTS: In response to SAFN stimulation at 10 Hz (n=7), a significant increase in BP (117.6 ± 7.5 %) occurred during the intra-stimulation period, while changes in CA (80.8 ± 7.4 %) and ICI (70.8 ± 17.2 %) were observed during the post-stimulation period. In 5 of 7 stimulation trials, we report episodes of bladder atonicity that lasted 33.0 ± 11.3 minutes. In response to 20 Hz SAFN stimulation (n=7), significant increases in BP (126.1 ± 13.3 %) and decreases in CA (79.9 ± 10.5 %) were observed, but the duration of bladder atonicity were markedly shorter (4.0 ± 1.2 min, 4 of 7 stimulation trials).
CONCLUSION: Our results show that the continuous urodynamic model can be used to measure the inhibitory effects of long-duration SAFN stimulation. During stimulation trials that resulted in bladder atonicity, we were able to measure increases in BP and decreases in the CA. With regards to eliciting an atonic bladder, further work is needed to clarify the effects of SAFN stimulation on voiding function.
Financial support for this project was provided by University of Toronto Connaught Fund; Canada Foundation for Innovation; and Canadian Institutes of Health Research (CIHR).
|10:09 a.m. #8|
EXPRESSION PROFILING OF EXPERIMENTAL NEUROGENIC BLADDER REVEALS DECREASED BETA 3-AR EXPRESSION THAT CAN BE REVERSED BY INOSINE TREATMENT.
Bryan Sack MD¹, Mary Piper PhD², Justin Cotellessa BSc³, Claire Doyle PhD⁴, Mehrnaz Gharaee-Kermani PhD, DVM³, Amy Avery BSc³, Fabliha Mahmood BSc⁴, Vivian Cristofaro PhD⁵, Maryrose Sullivan PhD⁵, Jill Macoska PhD³ and Rosalyn Adam PhD⁴
¹Boston Children's Hospital & Harvard Medical School; ²Harvard T.H. Chan School of Public Health, Boston, MA; ³University of Massachusetts Boston, Boston, MA; ⁴Boston Children's Hospital & Harvard Medical School, Boston, MA; ⁵VA Boston Healthcare System, West Roxbury, MA & Harvard Medical School, Boston, MA
Presented by: Bryan Sack
Introduction and Objectives: Neurogenic detrusor overactivity (NDO) is a devastating consequence of suprasacral spinal cord injury (SCI). Prior studies in our group revealed significant improvement in NDO in rats receiving chronic, systemic administration of the purine nucleoside inosine. However the molecular pathways underlying improvement are incompletely defined. The objective of this study was to use unbiased expression profiling to assess time-dependent transcriptional changes following SCI and to determine how expression of specific genes was altered by inosine.
Methods: RNAseq analysis was performed on full thickness bladder tissue from rats 2, 8 or 16 weeks after mid-thoracic spinal cord transection or from age-matched, non-injured controls. Differential gene expression and gene pattern clustering were performed using DESeq2 and DEGreport, respectively. In a separate cohort of animals, inosine was administered daily for 6 weeks by intraperitoneal injection. In each case, validation of gene expression changes was performed using real time RT-PCR.
Results: 207, 1355, and 2493 differentially expressed genes (DEGs) were identified at the 2, 8, and 16 week timepoints, respectively, with significant enrichment for gene ontology terms associated with cytoskeletal remodeling, synapse organization, axon guidance and neuromuscular junction activity. Among the significant DEGs, the beta 3- adrenergic receptor (β3-AR) was downregulated following SCI, whereas the M3 muscarinic receptor remained unchanged, suggesting a potential imbalance between sympathetic and parasympathetic responses. Notably, inosine treatment increased β3-AR levels in bladder tissue from SCI rats ~5-fold compared to vehicle-treated controls, and also increased β3-AR protein levels. In contrast, M3 mRNA levels were unaffected by inosine treatment.
Conclusions: Expression profiling of bladder tissue following SCI reveals perturbations in a variety of physiologically relevant gene clusters including those associated with innervation and cytoskeletal remodeling. Decreased β3-AR levels may reduce responses to sympathetic activity in the bladder leading to the development of NDO following SCI, whereas β3-AR levels can be reversed by treatment with inosine.
Funding: R01 DK077195 (RMA); T32 DK060442 (RMA).
|10:21 a.m. #9|QUANTIFICATION OF BLADDER WALL MICROMOTION DURING URODYNAMICS IN A NOVEL ANESTHETIZED PIG MODEL WITH LOW AMPLITUDE RHYTHMIC CONTRACTIONS USING M-MODE ULTRASOUND
Anna Nagle PhD¹, Zachary Cullingsworth BS², Uzoma Anele MD³, Charles Blocher MS³, Adam Klausner MD⁴ and John Speich PhD²
¹Department of Mechanical & Nuclear Engineering, Virginia Commonwealth University; ²Department of Mechanical & Nuclear Engineering, Virginia Commonwealth University, Richmond VA; ³Department of Surgery, Virginia Commonwealth University School of Medicine, Richmond, VA; ⁴Department of Surgery, Virginia Commonwealth University School of Medicine, Richmond, VA and Department of Surgery Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, VA
Presented by: Anna Nagle
Introduction and Objectives
Bladder wall micromotion caused by low amplitude rhythmic contractions (LARC) of the bladder wall may play a key role in producing bladder sensation and urgency. A non-invasive method to detect micromotion would be an important step in accessing conditions such as overactive bladder and detrusor overactivity. The aim of this study was to develop an anesthetized porcine model to compare LARC quantified from urodynamic pressures with micromotion quantified with non-invasive M(motion)-mode ultrasound cine loops of the bladder wall. The hypothesis of this study was that bladder wall micromotion measured with ultrasound represents a myogenic property that would directly correlate to LARC seen in vesical pressure (Pves).
Female pigs weighing 20-30 kg anesthetized with urethane underwent urodynamic studies with a fill rate of 50 ml/min. Expected cystometric capacity was 500 ml. At bladder volumes of 250 ml and 500 ml, filling was paused and ventilation was paused for 60 sec so that image data could be obtained without respiratory or filling motion. A correlation-based motion tracking algorithm was implemented in MATLAB to measure the width of the bladder wall over time in user-selected regions of interest (ROIs). The changes in bladder wall width were compared to changes in (Pves) via Pearson correlation coefficient.
Figure 1 shows a frame of an ultrasound M-mode cine loop in which bladder wall thicknesses were tracked in two ROIs (A) in the anterior bladder wall (blue) and the posterior bladder wall (magenta). Also shown are the changes in bladder width within those ROIs overlaid on Pves (B). The correlation between anterior and posterior bladder wall width compared with Pves were 0.61 and 0.62 respectively (p<0.001).
A novel anesthetized pig model exhibiting LARC was developed. A moderate correlation was found between bladder wall width and Pves. This shows proof of concept for measuring of bladder wall micromotion non-invasively with ultrasound. Further analysis of changes in bladder wall at different levels of bladder filling with and without rhythmic changes in Pves will increase understanding of the relationship between micromotion and LARC.
|10:33 a.m. #10|URINARY LEVELS OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1) PREDICT THE SEVERITY OF SYMPTOM AND RESPONSE TO TREATMENT IN PATIENTS WITH OVERACTIVE BLADDER (OAB)
Gamal Ghoniem MD FACS¹, Bilal Farhan MD², Ahmed Ahmed MD³ and Frank Zaldivar MD PhD³
¹UC,Irivne, CA; ²University of California, Irvine, CA; ³UC, Irvine, CA
Presented by: Bilal Farhan
INTRODUCTION AND HYPOTHESIS: We hypothesize that MCP-1 urinary levels correlate with OAB patients’ symptom severity. Our aim is to correlate normalized MCP-1 urinary levels to OAB symptoms before and after treatment. We conducted prospective study on patients with OAB symptoms and age-matched controlled
PATIENTS AND METHODS: Urinary MCP-1 levels were measured in 36 patients with OAB and 13 controls. Patients were treated after the first visit by different OAB treatments (anticholinergic, Beta-3 agonist and or, onabotulinum toxin A, neuromodulations). Urinary MCP-1 levels were measured by (ELISA). The urinary MCP-1 levels and OAB symptoms severity were compared at baseline, 1 month, and 3 months after treatments. Different validated OAB questionnaires were used.
RESULTS: The baseline urinary MCP-1 levels of patients with untreated OAB were significantly higher than that of controls with means. Urinary MCP-1 levels were significantly reduced at 3 months in 28 OAB-responders (77.8%). On other hand, 8 OAB- non-responders, showed unchanged in urinary MCP-1(Table 1).The severity of OAB symptoms and QoL had significantly decreased with urinary MCP-1 levels OAB- responders at 1 and 3 months of OAB treatments(Table 2).
Conclusion: Urinary MCP-1 levels were significantly higher in patients with OAB than in the controls. Patients with OAB who responded to treatments had significantly reduced urinary MCP-1 levels in association with a decreased severity of OAB symptoms at 3 months.
These promising findings could help understanding the pathophysiology of OAB and neurophysiological signaling in the bladder function, identification of a potential marker, and/or developing new drug targets for treatment of patients suffering from OAB.
Table 1: Urinary MCP-1 levels in controls, OAB patients (responders, non-responders)
Urinary MCP-1 levels
SubjectsNo. Baseline(Pre-treatment) 3-Months(post treatment)P-Value
Controls13 51.02 ± 9.63
Responders28 (77.8%)257.13 ± 49 72.87±13 <0.001
Non-responder8 (22.2%)246.5± 45 244.37±32=0.207
10:45 a.m. - 11:00 a.m.
11:00 a.m. - 12:00 p.m.
Keynote Speaker: Non-Voiding Contractions Encode Essential Information on Urinary Bladder Fullness: Implications for Urinary Bladder Dysfunction
Mark T. Nelson, PhD
12:00 p.m. - 12:15 p.m.
2018 Basic Science Prize Essay Award Presentation and Top Podium Selection
Una J. Lee, MD, FPMRS
Zachary Cullingsworth, BS
| #1|AUTOMATED QUANTIFICATION OF LOW AMPLITUDE RHYTHMIC CONTRACTIONS (LARC) DURING URODYNAMICS: IDENTIFICATION OF A DETRUSOR OVERACTIVITY SUBGROUP?
Zachary Cullingsworth BS¹, Brooks Kelly BS², Nicholas Deebel BS², Andrew Colhoun MD², Anna Nagle PhD³, Adam Klausner MD² and John Speich PhD³
¹Virginia Commonwealth University; ²Department of Surgery/Division of Urology, Virginia Commonwealth University, Richmond, Virginia; ³Department of Mechanical and Nuclear Engineering, Virginia Commonwealth University, Richmond, Virginia
Presented by: Zachary Cullingsworth
INTRODUCTION/OBJECTIVES: Low amplitude rhythmic contractions (LARC) of detrusor muscle may contribute to detrusor overactivity (DO) in some patients. The aim of this study was to develop an objective method to quantify LARC in urodynamics (UD) data and determine whether significant LARC correlated with a subgroup of patients with DO.
METHODS: An automated Fast Fourier Transform (FFT) analysis algorithm was developed to analyze a 205-second region of interest (ROI) of UD data ending 30 seconds before voiding (Fig 1A). The algorithm then identifies three frequencies in the 1.75-6 cycle/minute range (Fig 1B, shaded region) associated with the largest rhythmic amplitude peaks in vesical pressure (Pves). Peak Pves amplitudes (Fig 1B, X symbols) were analyzed to determine whether any significant rhythmic activity was present in Pves and if that activity was independent of any rhythm in abdominal pressure (Pabd). This algorithm was used for an Institutional Review Board-approved retrospective analysis of 98 UD studies.
RESULTS: During a blinded analysis, a neurourologist/urodynamicist identified 53/98 patients as having DO based on the UD pressure data. The FFT algorithm identified significant and independent (S&I) LARC in 19/53 patients with DO and 1/45 patients without DO, resulting in a specificity of 0.98, a sensitivity of 0.36 and a significant association (Fischer’s exact test, p<0.0001). The average slowest S&I LARC frequency was 2.81±0.22 cycles/min. Individual UD studies can be analyzed in under 5 seconds, allowing real-time interpretation.
CONCLUSIONS: Analysis of LARC during UD testing identified a subgroup of patients with DO with a distinct LARC frequency in Pves independent of Pabd. Refinements of this technique may identify subgroups of individuals with LARC−associated DO.
12:15 p.m. - 1:15 p.m.
Location: Austin Grand Ballroom Foyer, 6th Floor
1:15 p.m. - 2:50 p.m.
Panel 3: The Use of Stem Cells in Lower Urinary Tract Research*
Adonis Kheazee Hijaz, MD
Functional Tissue Reconstruction by an Acellular Regenerative Medicine Approach
Stephen F. Badylak, DVM, PhD, MD
Urothelial Progenitor Cells in Regeneration
Cathy L. Mendelsohn, PhD
Tissue Injury, Cytokine Response and Stem Cell Mobilization
Marc Steven Penn, MD, PhD, FACC
Stem Cells Modulate the Host Response to Urethral Injury
Adonis Kheazee Hijaz, MD
1:30 p.m. - 5:45 p.m.
Location: Room 616 AB, 6th Floor
4:00 p.m. - 6:30 p.m.
Fellowship Program Directors Meeting
Location: Room 415 AB, 4th Floor
2:50 p.m. - 3:00 p.m.
3:00 p.m. - 4:55 p.m.
Panel 4: NIH-Funded Flagship (O'Brien) Research Centers in Benign Urology: What Can the Core Facilities Offer to the Urology Research Community
Toby C. Chai, MD
Maryrose P. Sullivan, PhD
Urological Research Resources from the University of Pittsburgh
Urological Research Resources from the University of Wisconsin
William Ricke, PhD
Urological Research Resources from Columbia University
Cathy L. Mendelsohn, PhD
Urological Research Resources from Harvard University
Mark L. Zeidel, MD, MS
4:55 p.m. - 5:10 p.m.
5:10 p.m. - 7:00 p.m.
Basic Science Poster Session II
Lori A. Birder, PhD
Christopher John Chermansky, MD
CORRELATION BETWEEN DETRUSOR AND MOTOR FUNCTION IN AN ANIMAL MODEL OF PARKINSON’S DISEASE
Vivian Cristofaro PhD¹,², Andrew Orlando ¹,³, Sean D Carey ¹,³, Yifei Xu ¹,³, Josephine A Carew ¹,³ and Maryrose P Sullivan ¹,³
¹VA Boston Healthcare System, Harvard Medical School; ²Boston MA.; ³Boston MA
Presented by: Vivian Cristofaro
Introduction: Recent findings in patients with Parkinson’s disease (PD) indicate that symptoms associated with disorders of visceral organs, including the bladder, may precede by decades the onset of motor deficits. Abnormal aggregation of α-synuclein (α-syn) in the substantia nigra is a hallmark of PD. This protein has been implicated in synaptic vesicle trafficking. Although well characterized in the central nervous system, the expression and distribution of α-syn in peripheral organs has not been defined. This study used an animal model of PD to localize α-syn in the bladder, and investigate age-dependent detrusor abnormalities in relation to the progression of motor deficits.
Methods: α-syn localization was analyzed in normal bladder tissue by immunofluorescence microscopy. Bladders for functional studies were obtained from transgenic mice overexpressing a mutation of α-syn (SNCAA53T) that is common in human familial PD and from transgenic mice overexpressing the human α-syn wild-type (SNCAWT). Neurogenic responses to electrical field stimulation (EFS) were tested in bladders from 14, 28, 42, 58 week-old mice. Muscarinic M3 and purinergic P2X1 receptor expression in bladder tissue was determined by western blotting. Changes in motor function were evaluated by the rotarod test.
Results: α-syn immunoreactivity was detected throughout the bladder of normal mice and co-localized with the cholinergic marker VAChT. Motor performance was similar in both transgenic strains at 14 and 28 weeks. Compared to SNCAWT, coordination was impaired in SNCAA53T mice at 42wks and worsened further at 58wks. In bladder tissue, neurogenic contractions induced by EFS were similar between both transgenic 14wk old mice. However, by 28wks of age, EFS responses were higher in the mutant transgenic SNCAA53T mice compared to SNCAWT. At 42wks, EFS-induced responses were decreased in SNCAA53T mice and by 58 wks of age, were lower than responses in SNCAWT. Changes in M3R or P2X1R expression were not detected in these bladder tissues.
Conclusions: In this animal model of PD, changes in neurogenic detrusor contractions preceded motor dysfunctions, suggesting that altered peripheral innervation occurs at an early stage of PD. α-syn localization on excitatory nerves is consistent with its potential role in peripheral neurotransmission in the bladder. Transgenic mice overexpressing a human SNCA mutation are a promising model of bladder dysfunction in PD.
Funding: Dept. Veteran Affairs
SEARCHING FOR THE SOURCE: MACROSCOPIC MEASUREMENT OF CALCIUM SIGNALS AND MICROMOTIONS IN THE MOUSE URINARY BLADDER
Nathan Tykocki PhD, Grant Hennig PhD and Mark Nelson PhD
University of Vermont, Burlington, VT
Presented by: Nathan Tykocki
Multiple species, including humans, exhibit “non-voiding” or “transient” contractions (TCs) of the bladder wall during filling, that are accompanied by large bursts of activity from bladder sensory nerves. This suggests that TCs play an important role as sensors of bladder fullness, but the nature of TCs themselves remains unclear. Traditional confocal and multiphoton microscopy are limited by their reliance on high-power objective lenses, thin focal plane, and sensitivity to movement. This makes them unsuitable for measuring calcium signals and micromotions across the entire detrusor muscle syncytium, especially during a TC. Thus, we sought to devise a means of capturing and analyzing the macroscopic patterns of calcium signals and micromotions from the entire bladder wall, to determine how and where TCs originate. We designed and fabricated a specialized chamber to image bladder micromotions while simultaneously measuring intravesical pressure in an ex vivo mouse bladder. We discovered that a “TC” is the integration of multiple microcontractions of discrete portions of the bladder wall. Interestingly, these micromotions vary in frequency and duration, but result in regular, phasic increases in intravesical pressure characteristic of a TC. While calcium transients within the bladder smooth muscle seem stochastic in nature, global synchronous calcium events can and do occur. These techniques, while still in their infancy, represent new and novel tools to finally determine the nature and origin of TCs in the urinary bladder. Funded by NIH K01-DK103840 (NRT) and R37-DK053832 (MTN).
CHANGES IN DETRUSOR FUNCTION AND PROTEIN O-GLCNACYLATION IN AN ANIMAL MODEL OF TYPE 2 DIABETES
Yifei Xu ¹,², Josephine A Carew PhD¹,², Raj K Goyal MD¹,², Maryrose P Sullivan PhD¹,² and Vivian Cristofaro PhD¹,²
¹VA Boston Healthcare System, Harvard Medical School; ²Boston MA.
Presented by: Vivian Cristofaro
Introduction/Objectives: Diabetes is associated with a high prevalence of lower urinary tract symptoms. Although the time course of diabetic bladder dysfunction is well established in type-1 diabetes, temporal changes in bladder function in type-2 diabetes (T2D) are less clear. Chronic hyperglycemia increases protein O-GlcNAcylation (modification of serine/threonine residues by the moiety, N-acetyl-glucosamine) thus altering the activity of a variety of proteins. Using bladders from an animal model of T2D, this study compared detrusor function and protein O-GlcNAcylation patterns to investigate whether modifications of specific proteins might be responsible for altered detrusor function.
Methods: Bladders were procured from db/db and control mice at ages between 10 and 40 weeks. After removing the mucosa, longitudinal bladder smooth muscle (BSM) tissue was mounted in organ baths containing Kreb’s for isometric tension studies. BSM responses to nerve- or agonist-induced stimulations were compared between diabetic and control animals. Changes in protein GlcNAcylation were determined by western blotting of BSM tissue lysates from diabetic and control mice.
Results obtained: Contractions induced by EFS were significantly lower in db/db mice than in control tissue at 10 and 20 wks, but no differences were detected between groups at 30 or 40 wks of age. Post-junctional bladder contractions elicited by CCh and KCl were comparable between groups. A significant age-dependent increase in O-GlcNAc immunoreactivity was detected in db/db bladders compared with control. Akt, a kinase whose activation downstream of insulin signaling has been linked to vesicle exocytosis, was identified as highly O-GlcNAcylated in db/db BSM tissue.
Conclusions: The decreased neurogenic responses observed in db/db bladders together with the unchanged post-junctional excitatory responses compared to controls suggest impaired neurotransmission with early T2D. An increase in Akt O-GlcNAcylation may provide a molecular mechanism for the impaired neurotransmission in db/db bladders.
Funding: Dept. Veteran Affairs.
|Poster #BS21|APPLICATION OF NEAR INFRARED SPECTROSCOPY TO CHARACTERIZE HEMODYNAMICS OF PELVIC FLOOR MUSCULATURE IN WOMEN WITH LOWER URINARY TRACT SYMPTOMS AND VOIDING DYSFUNCTION
Emily Deegan BA, BScN, RN¹, Lynn Stothers D , FRCSC², Darren Lazare BEng, BA, MD, FRCSC³ and Andrew Macnab MD (London), FRCPC, FRCPCH, FCAHS⁴
¹Department of Experimental Medicine, University of British Columbia International Collaboration on Repair Discoveries; ²Department of Urological Sciences & Peter Wall Institute for Advanced Studies, University of British Columbia, International Collaboration on Repair Discovery (ICORD), Vancouver, British Columbia; ³Department of Obstetrics and Gynaecology & Department of Physical Therapy, University of British Columbia, BC Women’s Hospital, Vancouver, British Columbia; ⁴Stellenbosch Institute for Advanced Study, Wallenberg Research Centre, Stellenbosch, South Africa, Department of Urologic Sciences, University of British Columbia & International Collaboration on Repair Discoveries (ICORD), Vancouver, British Columbia
(Presented by: Emily Deegan)
I:Near infrared spectroscopy(NIRS) allows real time noninvasive measurement of muscle hemodynamics. It detects concentration of chromophores oxygenated(O2Hb) and deoxygenated hemoglobin(HHb) from which changes in blood volume and oxygen supply/demand are derived, allowing physiologic parameters of muscle function to be quantified. Assessment of pelvic floor muscle(PFM) function is central to managing urinary incontinence(UI) / lower urinary tract symptoms(LUTS) but currently lacks quantifiable physiologic measures. Objective:develop NIRS transvaginal probe to assess O2Hb and HHb in bilateral PFM and compare NIRS parameters in continent controls vs cases with LUTS due to neurogenic condition.
M:NIRS transvaginal probe incorporated fiberoptic cables in disposable optically inert plastic speculum. NIR light transmitted at 766,861,906 and 971nm by 2 channel Oxymon MkIII(Artinis Medical Systems). Paired optodes secured bilaterally at 20mm distance for 10mm tissue penetration. Data captured at 10Hz included O2Hb, HHb and totalHb tracings of right and left side. Subjects performed standardized PFM contractions with NIRS probe and perineometer, 10-min rest in between.
R:N=11, 5 neurogenic cases, Age 24-72yrs, BMI 20.2-26.9, Parity 0-3. Cause of neurogenic condition: spinal cord injury, multiple sclerosis, post-polio syndrome. To characterize hemodynamic pattern of PFM in response to exercise, O2Hb, HHb and tHb concentration changes were assessed. Changes arising with contraction are shown in Figure 1. Vertical bars=contraction,Y-Axis=[hemoglobin](-20 to 20 µmol),X-Axis=5 second intervals,red=O2Hb,blue=HHb,green=tHb. Asymmetry is demonstrated in physiologic response across bilateral PFM, even in controls. NIRS tracing decrease represents oxygen uptake required for muscular contraction. With neurogenic cases increased oxygen supply to tissue in less effected side occurs but lack uptake. This demonstrates a physiological response despite inability to produce contraction detectable by perineometer.
C:This pilot study demonstrates the feasibility of transvaginal NIRS probe to measure novel physiologic data in the context of PFM exercise and provides new insight on how symmetry and coordination may contribute to PFM function.
THE EFFECTS OF ACUTE ISCHEMIA ON INTRAVESICAL PRESSURE IN AN ISOLATED PERFUSED WORKING BLADDER MODEL
Andrew Tracey MD¹, Uzoma Anele MD¹, Andrew Colhoun MD², John Speich PhD³, Adam Klausner MD¹ and Paul Ratz PhD⁴
¹Virginia Commonwealth University Medical Center, Richmond, VA; ²Virginia Urology, Richmond, VA; ³Virginia Commonwealth University Department of Engineering, Richmond, VA; ⁴Virginia Commonwealth University School of Medicine, Richmond, VA
Presented by: Uzoma Anele
Chronic ischemia has been shown to negatively impact bladder function, but the effects of acute ischemia have not been fully elucidated. The present study used an isolated perfused working whole pig bladder model in combination with pig and human detrusor smooth muscle strips to examine the relationships between transient ischemia and bladder function.
Detrusor smooth muscle (DSM) strips were cut from fresh pig bladders obtained at a local slaughterhouse and from human cystectomy specimens. These strips were stretched and contracted at a standardized length with forces recorded in the setting of tissue starvation/hypoxia. In the whole pig bladder preparation, the vesical arteries were cannulated and perfused with physiologic buffer solution. A urethral catheter allowed filling at 40cc/min to a volume of 510cc, and bladders were then treated with intravascular carbachol or high-potassium superfusate to induce an isovolumetric contraction and void. A pressure sensor in the ureter continuously monitored vesical pressure during filling and contraction.
When exposed to 120 minutes of starved/hypoxic conditions, both pig and human DSM strips demonstrated significant increase in resting tone, with a greater than two-fold increase in force over control seen in pig strips (n=7, p<0.05), and a three-fold increase in resting force in human strips (n=2). Exposure to atropine completely blocked this increase in resting tone seen during starvation. Starved DSM strips also showed a significantly weaker contraction, but nearly a full recovery of contractile force after 15 minutes in a fed/oxygenated buffer.
In the whole bladder preparation, when filling occurred under ischemic conditions, the end-fill vesical pressure was significantly elevated over control (n=3, p<0.025), with a 225% increase in filling pressure. Subsequent fill-void cycle with normal perfusate flow (non-ischemic conditions) showed a return to baseline end-fill vesical pressure, not significantly elevated over control (n=3, p=0.43). Preliminary whole bladder data also suggests that exposure to atropine blocks the ischemia-induced rise in filling pressure.
Transient ischemia leads to an acute increase in tone in the working bladder model, thus reducing compliance. Furthermore, this increased contractility is reversible with re-perfusion and may be blocked with anticholinergics, suggesting a relationship between acute ischemia and increased local acetylcholine release.
|Poster #BS23|THE NATURAL HISTORY OF RADIATION CYSTITIS IN A RAT MODEL OF ACUTE AND CHRONIC LOWER URINARY TRACT DYSFUNCTION
Amy D. Dobberfuhl MD¹, Mason A. Briggs BS², Yan Wen MD², Shoucheng Ning PhD³, Edward E. Graves PhD³, Edward C. Diaz MD¹ and Bertha Chen MD²
¹Stanford University, Dept. of Urology; ²Stanford University, Dept. of Obstetrics and Gynecology; ³Stanford University, Dept. of Radiation Oncology
Presented by: Amy D. Dobberfuhl
Pelvic malignancy accounts for a third of new cancer cases and up to half receive radiation. Little is known about the natural history of radiation induced bladder dysfunction in rats. Our aim was to evaluate changes in lower urinary tract function over a 4-month period after radiation.
Thirty-six adult female Sprague Dawley rats were divided into 3 groups. Bladder was identified by computed tomography and irradiated on day 0 (0 Gy n=7; 20 Gy n=25; 30 Gy n=4). Void frequency and volume were recorded in 24 hour intervals using metabolic cages weekly (day 0-123). Bladders were assessed by cystometry and organ bath. Data were analyzed in SAS.
There were 6,078 ambulatory voids, representing 362 cage cycles and 16 time points. Four rats died from radiation proctitis. On Spearman analysis (0, 20, 30 Gy; n=4/group), void volume correlated with food intake (r=-0.41, p<0.01), rat weight (r=0.27, p<0.01) and stool output (r=-0.58, p<0.01). Meanwhile overnight urine output (r=0.05, p=0.54) and water intake (r=0.03, p=0.74) were independent of void volume. Using a mixed effect model to evaluate within and between group differences over time (Figure), there was a significant decrease in mean void volume after 20 Gy (p=0.014). High morbidity (n=2) and no recovery in void volume was noted in the 30 Gy group. Significant reduction in void volume and increase in frequency occurred during the first 30 days after radiation. At 3 months, recovery of void volume was noted only after 20 Gy. On cystometry (n=32) radiation resulted in a clear inverse relationship, where elevated threshold pressure correlated with loss of voiding efficiency. Radiation induced two distinct phenotypes at a threshold of 10% voiding efficiency. Small capacity end-stage bladders demonstrated high pressure (>40 cmH2O) uninhibited contractions, consistent with the chronic phase of bladder dysfunction. Whereas underactive bladders demonstrated high threshold pressure (>20 cmH2O), weak contractile amplitude (<10 cmH2O) and elevated post void residual (>700 uL).
After bladder radiation, acute dysfunction subsided by 1 month and there were two distinct phenotypes of chronic dysfunction noted at 3 months which were reflective of the human condition.
CHRONIC MEALTIME SHIFT DISTURBS METABOLIC AND URINARY FUNCTIONS IN MICE: EFFECTS OF DAILY SUPPLEMENTATION OF ANTIOXIDANTS
Kyung-Jin Chung MDPhD¹, Su Jin Kim ², Sung Tae Cho MDPhD³, Hyeong Gon Kim MDPhD⁴, Kyu-Sung Lee MDPhD⁵, Myung-Soo Choo MDPhD⁶, Khae Hawn Kim MDPhD⁷, Young-Suk Lee MDPhD8 and Jae Yup Hong MDPhD9
¹Department of Urology, Gachon University Gil Hospital, Gachon University of Medicine and Science, Incheon, Korea; ²Department of Urology, Seoul St. Mary’s Hospital, The Catholic University of Korea College of Medicine; ³Department of Urology, Hallym University Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea; ⁴Department of Urology, Konkuk University School of Medicine, Seoul, Korea; ⁵Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; ⁶Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea; ⁷Department of Urology, Gachon University Gil Medical Center, Gachon University School of Medicine, Incheon, Korea; 8Department of Urology, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine; 9Department of Urology, Cha University School of Medicine
Presented by: Su Jin Kim
INTRODUCTION: Local circadian clocks are present in three bladder tissues and lumbar spinal cord and mice defective in circadian clock genes exhibit alterations in water intake and excretion rhythms, suggesting functional clock-dependent nature of micturition functions. Yet, the effects of chronic circadian disturbance on urinary functions and their precise mechanisms are yet unclear. Therefore, we examined the effects of chronic mealtime shift on circadian patterns of food intake, water consumption and urinary excretion in young adult male mice.
METHODS: Normal and Per2::Luc knock-in mice were used. Water intake and urine output according to the circadian rhythm was checked using metabolic cage in 12:12 LD photoperiodic cycle and chronic mealtime shift. Circadian clock gene expression rhythm in bladder was evaluated with the activation of Per2 promotor. Per2 promoter activity in the bladder ex vivo from Per2::Luc Knock-in mice and reactive oxygen species (ROS) levels within the body were analysed. Circadian patterns of water intake and urinary excretion and the patterns of Per2 oscillation in the bladder were analyzed after antioxidants with melatonin or C3G.
RESULTS: Circadian patterns of water intake and urinary excretion were significantly affected by mealtime shift. Chronic mealtime shift increased the amplitude of Per2 oscillation in the bladder. In addition, mealtime shift clearly delayed its acrophase by delaying several hours. Mealtime shift induces imbalances between anti-oxidative capacity and ROS levels within the body, indicating increased oxidative damages during the rest phase in mice. Daily supplementation of antioxidants such as melatonin or C3G at ZT23 could block the insulin resistance caused by chronic mealtime shift. However, supplementation of antioxidants neither affected the mealtime shift-induced circadian patterns of water intake and urinary excretion, nor the altered patterns of Per2 oscillation in the bladder cultured ex vivo.
CONCLUSIONS: From these results, we suggest that chronic mealtime shift causes metabolic disturbances and urinary alterations via distinct separable mechanisms.
COMPARISON OF DETRUSOR ULTRASTRUCTURE IN WOMEN AND MEN WITH BLADDER OUTLET OBSTRUCTION – A POTENTIAL ROLE FOR DIAGNOSTIC BLADDER BIOPSY.
Audrey Wang MBBS, FRACS¹, Susan Brammah MApplSc², Amanda Chung MBBS, MS, FRACS³, Vincent Tse MBBS, MS, FRACS⁴ and Lewis Chan MBBS, FRACS, DDU⁴
¹Department of Urology, Westmead Hospital, Westmead NSW, Australia; ²Electron Microscopy Unit, Department of Anatomical Pathology, Concord Repatriation General Hospital, Concord NSW, Australia; ³The University of Sydney, Sydney Medical School, Department of Urology, Concord Repatriation General Hospital, Concord NSW, Australia.; ⁴The University of Sydney, Sydney Medical School, Department of Urology, Concord Repatriation General Hospital, Concord NSW, Australia
Presented by: Amanda Chung
Introduction and Objectives:
Previous ultrastructural studies in men with bladder outlet obstruction (BOO) have demonstrated that features of myohypertrophy (muscle fascicle derangement, collagenosis, variation in myocyte size/shape) were associated with worse voiding outcomes following transurethral resection of the prostate. The objective of this study was to compare ultrastructural features using the same standardised protocol in female and male patients with BOO to assess whether detrusor biopsy may have a role in the diagnosis of female BOO.
Thirteen patients (7 female, 4 male) with known BOO on urodynamic study and 2 control patients (female) with normal urodynamic studies underwent cystoscopy and detrusor muscle biopsy. The detrusor muscle biopsy specimens were processed for transmission electron microscopy. Previously established diagnostic criteria were used for ultrastructural analysis (eg muscle fascicle derangement, myocyte irregularity, myocyte cell separation, collagenosis, cellular degeneration). The severity of the ‘myohypertrophy pattern’ was assessed and correlated with clinical features.
Features of myohypertrophy (muscle fascicle derangement, collagenosis, variation in myocyte size/shape) were present in the bladder specimens of all female and male patients with BOO but absent in the bladder specimens of control patients. Features of degeneration were present in varying degrees in the bladder specimens of all patients, consistent with previous studies showing that degeneration correlates with age rather than degree of obstruction. Myohypertrophy features were less marked in females with BOO compared to males with BOO, except in one female with prolonged voiding dysfunction after colposuspension. Myohypertrophy features were also seen in one patient with 4 months history of an obstructive sling. Semi-quantitative analysis of ultrastructural features showed severity of myohypertrophy correlated with duration and degree of obstruction in female BOO.
We have demonstrated similar ultrastructural features using a standardized protocol in detrusor biopsies of female patients with BOO compared to male BOO. In particular the myohypertrophy pattern is present in female BOO and is less marked than male BOO but appears to correlate with duration and severity of obstruction. Given the uncertainty in diagnosis of female BOO on urodynamic parameters, the detrusor biopsy may have a potential role in the diagnosis of female BOO.
PROPHYLACTIC TREATMENT MARKEDLY IMPROVES BLADDER CAPACITY FOLLOWING PELVIC RADIATION
Doreen Chang MA¹, Bryce A. Allio MD², Jillene M. Brooks MS³, Danielle J. Degoski BS³, A. Adam Kahokehr MD PhD², Andrew C. Peterson MD² and Matthew O. Fraser PhD⁴
¹Duke University School of Medicine, Duke University Medical Center, Durham, NC; ²Division of Urology, Department of Surgery, Duke University Medical Center, Durham, NC; ³Institute for Medical Research, Durham, NC; ⁴Division of Urology, Department of Surgery, Duke University Medical Center and Department of Research and Development, Durham Veterans Affairs Medical Center, Durham, NC
Presented by: Matthew O. Fraser
Introduction and Objectives: Radiation cystitis (RC) occurs frequently following therapeutic radiotherapy for pelvic malignancy. The acute phase of RC may be due to loss of urothelial barrier function. In some patients, the late phase of RC has devastating health outcomes. We hypothesize that the acute phase contributes or is directly responsible for the late phase. Utilizing our previously established animal model, we tested a novel therapeutic strategy to prevent the acute phase of RC.
Methods: Seven female Sprague Dawley rats received pre-radiation treatment with triamcinolone acetonide, 200 mg/ml in 50% dimethyl sulfoxide (DMSO) solution, 0.5 ml instilled into the bladder via the indwelling bladder catheter, while 10 served as controls with normal saline instillation. All animals received single dose bladder irradiation with 20 Gy using a CT image guided irradiator while retaining instillates. Bladder function was evaluated on a weekly basis for all animals for 9 weeks by conscious restrained cystometry using sequential infusions of normal saline, 300 and 500 mM KCl (≥ 60 minutes for each infusate; 0.10 ml/min flow rate). Cystometric functional and total bladder capacities (FBC, TBC) were recorded. Data were analyzed using 2−Way ANOVA and linear regression models with post-hoc testing.
Results: Prophylactic treatment resulted in marked improved in mean TBC from week 3 onward as follows: up to 64% greater following saline challenge (P=0.0008), up to 72% greater following 300KCl challenge (P=0.0021), and up to 91% greater following 500 KCl challenge (P=0.0001). Similarly mean FBC was consistently higher from 3 week onward as follows: by up to 38% greater following saline challenge (P=0.0034), up to 37% greater following 300KCl challenge (P=0.003), up to 49% greater following 500 KCl challenge (P<0.0001). Figure shows mean TBC and FBC.
Conclusions: These data demonstrate that prophylactic treatment with triamcinolone acetonide in DMSO solution results in improved cystometric profile in irradiated rats when compared to controls. These results may be translatable into future therapies to prevent RC in patients undergoing pelvic radiotherapy.
Financial Funding: Duke Urology
INDIVIDUALIZED ADD-ON TREATMENT BASED ON THE DIFFERENCE OF RECEPTOR OF ALPHA BLOCKER IN ANIMAL MODELS OF OVERACTIVE BLADDER AND BENIGN PROSTATE HYPERPLASIA
Sung Tae Cho MD, PhD¹, Don Kyoung Choi MD¹, Ohseong Kwon MD¹, Khae Hawn Kim MD,PhD² and Ji-Yeon Han MD³
¹Hallym University Kangnam Sacred Heart Hospital, Seoul, Korea; ²Gachon University Gil Hospital; ³Department of Urology, Pusan National University Yangsan Hospital, Pusan, Korea
Presented by: Sung Tae Cho
Introduction: ɑ1-antagonists are widely used for the treatment of lower urinary tract symptoms (LUTS). However, add-on therapy using different ɑ1-antagonist has not yet been determined. The aim of this study was to investigate the efficacy and safety of add-on therapy using various ɑ1-antagonists in animal models of overactive bladder (OAB) and benign prostatic hyperplasia (BPH) through urodynamic evaluation and measurements of angiogenesis related factors.
Methods: Female SD rats were used in OAB study, while male SD rats were used in BPH study. OAB was induced by i.p. injection of cyclophosphamide (75 mg/kg) every third day for 10 days. The female rats were divided into six groups (n=10 in each group): control group, OAB group, alfuzosin-treated OAB group, naftopidil-treated OAB group, tamsulosin-treated OAB group and naftopidil with tamsulosin-treated OAB group. BPH was induced by bilateral orchiectomy and injection of testosterone (20mg/kg) 0.5ml s.c. The male rats were also divided into six groups (n=10 in each group): control group, BPH group, alfuzosin-treated BPH group, naftopidil-treated BPH group, tamsulosin-treated BPH group and naftopidil with tamsulosin-treated BPH group. The rats in the treated groups orally received drugs once a day for 14(OAB) and 30(BPH) consecutive days. Cystometry was performed in 14(OAB) and 30(BPH) days. After the cystometry the expression levels of VEGF, IGF-1 and TGF-β of the bladder and prostate were quantified by Western blotting.
Results: On cystometry, the single ɑ1-antagonist therapy showed more improved voiding function in OAB and BPH models than combined therapy in terms of contraction pressure and time. In addition, ɑ1-antagonists facilitated the recovery of tissues from injury caused in animals with OAB and BPH. Rats with OAB and BPH showed increased expressions in angiogenesis related factors including VEGF, IGF-1, and TGF-β. On the other hand, both single and combined ɑ1-antagonist therapies suppressed increases of angiogenetic factors in the bladder and prostate.
Conclusions: In the present study, single therapy using tamsulosin showed the best effect in urodynamics evaluation and measurement of factors using western blot. We expected that combined therapy would be better than single therapy due to various pharmacological properties. However, there was no superiority of combined therapy for treatment of OAB and BPH in this study.
|Poster #BS28|COMPARISON OF ANTERIOR VAGINAL WALL INDENTATION PARAMETERS IN AGE-MATCHED CONTROL AND PROLAPSE PATIENTS USING AN OPERATOR INDEPENDENT ARTIFICIAL FINGER
Connie Wang BA, Panos Shiakolas PhD¹, Michael Abraham BS¹, Christopher Abrego BS¹ and Philippe Zimmern MD²
¹UTA; ²UT Southwestern Medical Center
Presented by: Connie Wang
Introduction and Objectives: To compare reaction forces of the anterior vaginal wall in control (C) and prolapsed (P) women in response to pressure applied at different angles of indentation through an automated artificial finger equipped with a distal sensor.
Methods: Following IRB approval, a tripod-mounted, artificial finger equipped with a calibrated, piezoresistive sensor at its tip and automated by NI LabView 2015 software for motion control via an actuator was used to create anterior vaginal wall deformations at 10, 15 and 20 degree angles. Age-matched women in the C and P groups were compared. All measurements were performed in the supine position in the operating room, with patients under general anesthesia prior to the start of the operation and after the bladder was drained. Each deformation included a 1 second upwards indentation, a 1 second maintenance “hold”, and a 1 second return of the fingertip to the baseline. Measurements were done in triplicate with a 3 second interval between each deformation sequence. Real-time voltages, equivalent to reaction forces sensed by the sensor during each indentation, were modeled as function of motion profiles and analyzed in Excel (See Figure). The motion profile of each indentation was used to calculate baseline voltage, amplitude change over the 1 second interval of upwards indentation, and slope of the upwards indentation curve in its median 0.5 second range.
Results Obtained: Five women of similar age group (mean 64, 51-73) were studied in each group. A significant difference was observed between all degrees of indentation in baseline voltage in P and C groups (p<0.05). At 10 and 20 degrees of indentation, there was a significant difference in amplitude change between P and C groups, while there was a significant difference in slope of indentation at 15 degrees between P and C groups.
Conclusion: The biomechanical properties of the human anterior vaginal wall can be objectively determined by a new device resembling the human finger. This mounted, free-standing artificial finger can apply a predictable and reproducible deformation to the anterior vaginal wall to compare the indentation properties of vaginal tissue in prolapsed and non-prolapsed conditions.
|Poster #BS29|BETA-3 ADRENOCEPTOR EXPRESSION IN THE UTEROSACRAL LIGAMENT IN THE POSTMENOPAUSAL WOMEN WITH PELVIC ORGAN PROLAPSE
Woojin Chong MD¹, John Andrew Fantl MD¹, Michael Donovan MD, PhD² and Charles Ascher-Walsh MD¹
¹Division of Female Pelvic Medicine & Reconstructive Surgery. Department of Obstetrics, Gynecology and Reproductive Sciences. Mount Sinai Medical Center/Icahn School of Medicine, NY, NY; ²Department of Anatomic Pathology. Mount Sinai Medical Center/Icahn School of Medicine, NY, NY
Presented by: Woojin Chong
Introduction/Objectives: Pelvic organ prolapse (POP) affects over 50% of adult women. It is commonly associated with a variety of lower urinary tract symptoms like overactive bladder (OAB). A β3-adrenoceptor (β3-AR) agonist (mirabegron) has been used to manage OAB. It is known that β3-ARs are found in the bladder detrusor smooth muscle (SM). Stimulation of β3-AR relaxes the detrusor SM and consequently improves bladder compliance in OAB patients. If such receptors are present in the pelvic floor such as uterosacral ligaments (USLs), β3-AR agonist may also relax the SM in the pelvic floor. As a first step, this pilot/descriptive study is designed to investigate the presence of β3-AR expression in the USLs in the postmenopausal (PMP) women with POP. Methods: Ten PMP women with known POP were recruited and consented. A piece of the distal USL was collected unilaterally at the time of the surgery; placed immediately in freshly prepared medium; and transferred to the Pathology lab for further histologic evaluation. H&E and immunohistochemistry (IHC) were performed. Positive (human female bladder) and negative (omission of primary antibody) controls were run in parallel. Imaging was captured using a digital camera (Nikon Digital sight D5-Fi2). Percentages of SM cells & connective tissues in the USLs and presence/percentage of β3-AR expression in SM cells were subjectively measured by an experienced pathologist. The demographic variables were expressed as means ± SEM of the number of subjects. The staining results were expressed as descriptive analysis. Results: Under high power field, the USLs were mainly composed of SM cells (81.5% ±7.47) and connective tissues (16.5%±7.9). On IHC analysis, 6 out of 9 specimens expressed β3-AR in SM cells with different level of expression (1 specimen failed proper staining). Figure 1 presents H&E and IHC analysis. Conclusions: The majority of the distal USLs were composed of SM cells. β3-ARs are expressed in 67% of the USLs specimens from women with POP. Considering that both POP and OAB are frequently seen in the elderly population, the effect of β3-AR agonist on the pelvic floor tissues should be investigated further.
No financial disclosures.