PD55-02 Altered Toll-Like Receptor 4 Activation Plays a Critical Role in Cystitis Pain: A Mapp Research Network Animal Model Study

THE JOURNAL OF UROLOGYâ e1056 INTRODUCTION AND OBJECTIVES: Previous studies showed bladder afferent hyperexcitability in bladder pain syndrome/ interstitial cystitis (IC/BPS). The aim of current study is to investigate the urothelial neurotrophin and their receptors expression of IC/BPS patients. METHODS: The patients with IC/BPS who were admitted for cystoscopic hydrodistention were recruited. The symptoms severity was investigated with Interstitial Cystitis Symptoms and Problem Index (ICSI and ICPI), O0 Leary-Saint symptom score (OSS), Visual Analogue Scale for pain (VAS) and cystometric bladder capacity (CBC). Random cold-cup biopsies of the posterior bladder wall were obtained during the procedure. Western blotting with quantification was used to investigate the neurotrophin expression of growth associated protein 43 (GAP-43), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF). The urothelial tropomyosin receptor kinase A (TrkA, receptor of NGF) and Trk-B (receptor of BDNF) were also investigated. Bladder specimens from female patients with stress urinary incontinence were also obtained for western blotting and were considered as control subjects. RESULTS: Total 24 control subjects, 51 non-ulcer IC/BPS and 23 ulcer IC/BPS were enrolled. The patients with non-ulcer IC/BPS was significantly younger than ulcer IC/BPS and control subjects (p<0.001). The expression of urothelium NGF and Trk-A was significantly higher in ulcer IC/BPS than that in control and non-ulcer IC/BPS (Table 1). The urothelium BDNF expression in non-ulcer IC/BPS was significantly lower, and the Trk-B was also significantly lower in ulcer IC/BPS. Among the all IC/BPS patients, the urothelium GAP-43 expression was significantly correlated with ICSI (r¼-0.363, ¼0.007) and OSS (r¼-0.301, p¼0.027). The Trk-A was also correlated with ICSI (r¼0.372,p¼0.006), OSS (r¼0.316, p¼0.020), VAS (r¼0.331, p¼0.015) and CBC (r¼-0.315, p¼0.013). CONCLUSIONS: The urothelium NGF and Trk-A expression was elevated in the ulcer IC/BPS, while the urothelium Trk-B expression was decreased. The GAP-43 and Trk-A expression were correlated to clinical symptoms. The urothelium expression of neurotrophin and their receptors may involve the pathogenesis of IC/BPS. Vol. 199, No. 4S, Supplement, Monday, May 21, 2018 INTRODUCTION AND OBJECTIVES: Altered Toll-like receptor (TLR) activation has been identified in several chronic pain conditions but has not been well studied in interstitial cystitis/bladder pain syndrome (IC/BPS). Our prior human studies indicated altered TLR4mediated inflammatory responses in IC/BPS, which was significantly correlated with pain severity and the extent of comorbid pain in patients. The objective of this study was to determine whether altered TLR4 activation plays a role in pelvic/bladder pain seen in IC/BPS using our validated transgenic autoimmune cystitis model (URO-OVA). METHODS: TLR4-deficient URO-OVA (URO-OVA/TLR4-/-) mice were generated through crossbreeding of URO-OVA mice with TLR4-/- mice. Both URO-OVA and URO-OVA/TLR4-/- mice (female, 68 weeks old, and n¼10 per group) were induced for cystitis through adoptive transfer of antigen-specific CD8þ T cells. At day 7 after cystitis induction, mice were analyzed for pelvic and bladder pain using von Frey filament stimulation and bladder distention-evoked visceromotor response (VMR), respectively. To block TLR4 activation, URO-OVA mice were intravenously injected with TAK-242 (a small TLR4 antagonistic molecule) at 2.5 mg/kg (n¼10) or vehicle (n¼10) at day 7 after cystitis induction. Mice were analyzed for pelvic/bladder pain at one hour post-treatment. Splenocytes were prepared and stimulated with escalating doses of lipopolysaccharide (LPS, a TLR4 agonist) ranging 10-5-102 mg/ml for 24 hours to evaluate TLR4-mediated inflammatory responses, followed by ELISA analysis of proinflammatory cytokines (IL-1b, IL-6, and TNF-a) in the conditioned culture supernatants. RESULTS: After cystitis induction URO-OVA mice developed pelvic and bladder pain as well as increased splenocyte production of TLR4-mediated IL-1b, IL-6, and TNF-a. Compared to URO-OVA mice, URO-OVA/TLR4-/- mice exhibited significantly reduced pelvic/bladder pain after cystitis induction. Intravenous treatment with TAK-242 reduced splenocyte production of TLR4-mediated proinflammatory cytokines and pelvic/bladder pain in URO-OVA mice compared to vehicletreated mice. CONCLUSIONS: Altered TLR4 activation plays a critical role in pelvic/bladder pain in the URO-OVA model, providing a potential mechanistic insight and a therapeutic target for IC/BPS pain. Source of Funding: NIH U01DK082344 and R01DK100891 PD55-03 PAR4 ACTIVATION AS A PERSISTENT MODEL OF BLADDER PAIN: ROLE OF MIF AND HMGB1 Fei Ma*, David Hunt, Lexington, KY; Lin Leng, Richard Bucala, New Haven, CT; Katherine Meyer-Siegler, St. Petersburg, FL; Pedro Vera, Lexington, KY Source of Funding: none PD55-02 ALTERED TOLL-LIKE RECEPTOR 4 ACTIVATION PLAYS A CRITICAL ROLE IN CYSTITIS PAIN: A MAPP RESEARCH NETWORK ANIMAL MODEL STUDY Xiangrong Cui, Xuan Jing, Susan Lutgendorf, Catherine Bradley, Bradley Erickson, Vincent Magnotta, Iowa City, IA; Andrew Schrepf, Ann Arbor, MI; Karl Kreder, Michael O’Donnell, Yi Luo*, Iowa City, IA INTRODUCTION AND OBJECTIVES: Painful bladder syndrome/interstitial cystitis (PBS/IC) is characterized by bladder-related pain without other bladder pathology. Animal models that mimic symptoms of PBS/IC are used to explore the possible mechanisms of PBS/IC. We showed that activation of intravesical protease activated receptor 4 (PAR4) induced acute bladder pain without other signs of inflammation and this effect was mediated through macrophage migration inhibitory factor (MIF) and high mobility box group 1 (HMGB1). Urothelial HMGB1 release was increased upon PAR4 activation and MIF inhibition blocked HMGB1 release, indicating MIF mediates HMGB1 release in urothelium after PAR4 activation. We extended this model by using repeated PAR4 instillation to elicit persistent bladder pain and to allow a time window for treatment strategies. We hypothesized that MIF and HMGB1 also mediate persistent bladder pain.