Background Activation of intravesical protease activated receptor 4 (PAR4) leads to release of urothelial macrophage migration inhibitory factor (MIF). MIF then binds to urothelial MIF receptors to release urothelial high mobility group box-1 (HMGB1) and elicit bladder hyperalgesia. Since MIF binds to multiple receptors, we investigated the contribution of individual urothelial MIF receptors to PAR4-induced HMGB1 release in vivo and in vitro and bladder pain in vivo. Methodology/Principal findings We tested the effect of intravesical pre-treatment with individual MIF or MIF receptor (CD74, CXCR4, CXCR2) antagonists on PAR4-induced HMGB1 release in vivo (female C57/BL6 mice) and in vitro (primary human urothelial cells) and on PAR4-induced bladder hyperalgesia in vivo (mice). In mice, PAR4 induced HMGB1 release and bladder hyperalgesia through activation of intravesical MIF receptors, CD74 and CXCR4. CXCR2 was not involved in these effects. In primary urothelial cells, PAR4-induced HMGB1 release through activation of CD74 receptors. Micturition parameters in mice were not changed by any of the treatments. Conclusions/Significance Urothelial MIF receptors CD74 and CXCR4 mediate bladder pain through release of urothelial HMGB1. This mechanism may set up persistent pain loops in the bladder and warrants further investigation. Urothelial CD74 and CXCR4 may provide novel targets for interrupting bladder pain.

Activation of intravesical PAR4 receptors leads to bladder hyperalgesia (BHA) through release of urothelial macrophage migration inhibitory factor (MIF) and urothelial high mobility group box-1 (HMGB1). MIF deficiency and/or MIF antagonism at the bladder block BHA in mice yet the mechanisms are not clear. Since oxidative stress and ERK phosphorylation are involved in MIF signaling we hypothesized that oxidative stress and/or ERK signaling, activated by MIF release, promote intravesical HMGB1 release to induce BHA. We induced BHA by intravesical PAR4 infusion in female C57BL/6 mice. Mechanical sensitivity was evaluated by measuring abdominal von Frey (VF) 50% thresholds before (baseline) and 24 h post-infusion. Intravesical pre-treatment (10 min infusion prior to PAR4) with N-acetylcysteine amide (NACA; reactive-oxygen species scavenger; 3 mg in 50 μl), FR180204 (selective ERK1/2 inhibitor; 200 μg in 50 μl), ethyl pyruvate (EP; HMGB1 release inhibitor; 600 μg in 50 μl), or diluent controls (50 μl) tested the effects of pre-treatment on PAR4-induced BHA. Intravesical fluid was collected after each treatment and HMGB1 concentration was measured using ELISA. Awake micturition parameters (volume and frequency) were assessed at the end of the experiments. Bladders were collected and examined for histological signs of edema and inflammation. Pre-treatment with PBS followed by PAR4 induced BHA in mice but PBS followed by scrambled peptide did not. Pre-treatment with NACA or EP partially blocked PAR4-induced BHA while FR180204 had no effect. A significant correlation between intravesical HMGB1 levels and 50% VF thresholds was observed. All PAR4 treated groups had increased levels of HMGB1 in the intravesical fluid compared to PBS-Scrambled group although not statistically significant. No significant effects were noted on awake micturition volume, micturition frequency or histological evidence of bladder edema or inflammation. Our results show that intravesical antagonism of bladder reactive-oxygen species accumulation was effective in reducing PAR4-induced bladder pain. The correlation between intravesical levels of HMGB1 and bladder pain indicates that released HMGB1 is pivotal to bladder pain. Thus, modulating events in the MIF signaling cascade triggered by PAR4 activation (including bladder oxidative stress and HMGB1 release) warrant further investigation as possible therapeutic strategies.

Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.

INTRODUCTION AND OBJECTIVE:The Angiotensin is widely known for its roles in blood pressure and fluid homeostasis. Recently it has been implicated in several facets of metastatic bone disease including inflammation, angiogenesis, tumor cell proliferation, and migration. In addition, in a model of prostaglandin-induced hyperalgesia, Ang-(1-7) dose-dependently attenuated behavioral signs of pain. Aim:The study was undertaken to test the hypothesis that Mas receptor activation by specific agonist could be an effective therapeutic target to alleviate bladder pain syndrome and/or chronic pelvic pain.METHODS: Two groups of female rats: Mas receptor wild type (Mas R WT) and knockout (KO) were used in this study. The electromyographic (EMG) recording from the external oblique muscles (EOM) of the abdomen during urinary bladder distension (UBD) is a direct method for measuring pain sensation in awake rats. In these experiments, rats were anesthetized with 2% Isoflurane and a pair of Tefloncoated electrodes were chronically implanted into the EOM for EMG recordings. Three days after the surgery, before EMG recording of viscero-motor reflex (VMR) to UBD rats were lightly anesthetized with isoflurane and a polyethylene catheter (PE-50) was inserted transurethrally into the bladder and sealed with tissue glue to prevent voiding. This allowed us to generate intravesical pressure without spontaneous voiding, a model of obstruction pain. The bladder was distended with saline either by slow infusion (0.1ml/min) or graded phasic isobaric distensions (10, 20, 30, 40 and 60mmHg). The increasing EOM contractions to incrementing intravesical pressures was considered as cramping pain of the abdomen represented as VMR.RESULTS: The Mas R WT rats exhibited a threshold for VMR at distending pressure >20mmHg, whereas Mas R KO exhibits threshold <10mmHg and higher. The results suggest that Mas R KO rats are more sensitive to visceral sensation compared to WT rats (F Distension (5, 66) [ 132.3, p <0.0001). Mas R WT and KO rats injected with AVE0991, a Mas R agonist, abolished VMR to UBD in WT, but not in KO rats (F Distension (5, 66) [ 132.3, p <0.0001).CONCLUSIONS: Based on these data Mas R may play a role in visceral pain perception. The Ang (1-7)/Mas receptor axis could be an effective therapeutic target to alleviate visceral pain.

Basso, Beattie and Bresnahan (BBB) locomotor rating scale. Bladder tissue samples from controls ( 14) and spinal contused (2 and 9 weeks post-injury) were subjected to uniaxial stress relaxation at 50% strain to determine instantaneous and relaxation modulus, and monotonic load-to failure at 1% strain/sec to determine Young's modulus, yield stress and strain, and ultimate stress and strain.RESULTS: None of the rats had a normal BBB score. At 2 weeks post-injury, SCI vs control showed instantaneous and relaxation modulus were decreased 64.3% (p>0.99) and 70.6% (p[0.07), respectively. Similarly, at 9 weeks post-injury, instantaneous and relaxation modulus were decreased by 71.9% (p[0.04) and 71.4% (p[0.13), respectively, compared to controls. Young's modulus of SCI rats was decreased 50% (p>0.99) at 2 weeks and 49% (p>0.99) at 9 weeks compared to controls. Yield stress of SCI bladders was decreased 55% (p >0.99) compared to controls at 2 weeks post-injury but was increased 62% (p[0.027) relative to controls at 9 weeks. Yield strain showed no difference at 2 weeks post-injury but increased 90% (p<0.01) in SCI rats at 9 weeks post-injury. Ultimate stress was decreased 37% (p[0.39) at 2 weeks post-injury in SCI rats relative to control rats, but no differences were observed at 9 weeks post-injury, and no differences in ultimate strain were observed between groups at either time point.CONCLUSIONS: We found that the mechanical properties of rat bladder wall after a spinal cord injury at week 2 showed little differences compared to controls, but by week 9 SCI bladders had a greater yield stress and yield strain in the spinal cord injury bladders at 9 weeks compared to 2 weeks and control. This suggests that SCI rat bladders were more compliant than control counterparts.