Xiao-Qing Pan

Experimental colitis triggers the release of substance P and calcitonin gene-related peptide in the urinary bladder via TRPV1 signaling pathways.

Clinical data provide evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5mg/kg), and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10(-)(7)M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p ≤ 0.05) followed by 35-fold increase at day 30 (n=5, p ≤ 0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA 1 month after TNBS (n=5, p ≤ 0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p ≤ 0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p ≤ 0.05). Colitis did not alter CGRP concentration during acute phase; however, at day 30 mRNA level was increased by 17.8 ± 6.9-fold (n=5, p ≤ 0.05) in parallel with 4-fold increase in CGRP protein (n=5, p ≤ 0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45-65% (p ≤ 0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder; however, it caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization.

Differential effects of intravesical resiniferatoxin on excitability of bladder spinal neurons upon colon–bladder cross-sensitization

Cross-sensitization in the pelvis may contribute to etiology of functional pelvic pain disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increasing evidence suggests the involvement of transient receptor potential vanilloid 1 (TRPV1) receptors in the development of neurogenic inflammation in the pelvis and pelvic organ cross-sensitization. The objective of this study was to test the hypothesis that desensitization of TRPV1 receptors in the urinary bladder can minimize the effects of cross-sensitization induced by experimental colitis on excitability of bladder spinal neurons. Extracellular activity of bladder neurons was recorded in response to graded urinary bladder distension (UBD) in rats pretreated with intravesical resiniferatoxin (RTX, 10(-7)M). Colonic inflammation was induced by intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The duration of excitatory responses to noxious UBD during acute colonic inflammation (3 days post-TNBS) was significantly shortened in the group with RTX pretreatment (25.3±1.5s, n=49) when compared to the control group (35.1±4.2s, n=43, p<0.05). The duration of long-lasting excitatory responses, but not short-lasting responses of bladder spinal neurons during acute colitis was significantly reduced by RTX from 52.9±6.6s (n=21, vehicle group) to 34.4±2.1s (RTX group, n=21, p<0.05). However, activation of TRPV1 receptors in the urinary bladder prior to acute colitis increased the number of bladder neurons receiving input from large somatic fields from 22.7% to 58.2% (p<0.01). The results of our study provide evidence that intravesical RTX reduces the effects of viscerovisceral cross-talk induced by colonic inflammation on bladder spinal neurons. However, RTX enhances the responses of bladder neurons to somatic stimulation, thereby limiting its therapeutic potential.

Lack of transient receptor potential vanilloid 1 channel modulates the development of neurogenic bladder dysfunction induced by cross-sensitization in afferent pathways

BackgroundBladder pain of unknown etiology has been associated with co-morbid conditions and functional abnormalities in neighboring pelvic organs. Mechanisms underlying pain co-morbidities include cross-sensitization, which occurs predominantly via convergent neural pathways connecting distinct pelvic organs. Our previous results showed that colonic inflammation caused detrusor instability via activation of transient receptor potential vanilloid 1 (TRPV1) signaling pathways, therefore, we aimed to determine whether neurogenic bladder dysfunction can develop in the absence of TRPV1 receptors.MethodsAdult male C57BL/6 wild-type (WT) and TRPV1−/− (knockout) mice were used in this study. Colonic inflammation was induced by intracolonic trinitrobenzene sulfonic acid (TNBS). The effects of transient colitis on abdominal sensitivity and function of the urinary bladder were evaluated by cystometry, contractility and relaxation of detrusor smooth muscle (DSM) in vitro to various stimuli, gene and protein expression of voltage-gated sodium channels in bladder sensory neurons, and pelvic responses to mechanical stimulation.ResultsKnockout of TRPV1 gene did not eliminate the development of cross-sensitization between the colon and urinary bladder. However, TRPV1−/− mice had prolonged intermicturition interval and increased number of non-voiding contractions at baseline followed by reduced urodynamic responses during active colitis. Contractility of DSM was up-regulated in response to KCl in TRPV1−/− mice with inflamed colon. Application of Rho-kinase inhibitor caused relaxation of DSM in WT but not in TRPV1−/− mice during colonic inflammation. TRPV1−/− mice demonstrated blunted effects of TNBS-induced colitis on expression and function of voltage-gated sodium channels in bladder sensory neurons, and delayed development of abdominal hypersensitivity upon colon-bladder cross-talk in genetically modified animals.ConclusionsThe lack of TRPV1 receptors does not eliminate the development of cross-sensitization in the pelvis. However, the function of the urinary bladder significantly differs between WT and TRPV−/− mice especially upon development of colon-bladder cross-sensitization induced by transient colitis. Our results suggest that TRPV1 pathways may participate in the development of chronic pelvic pain co-morbidities in humans.

Response of the human detrusor to stretch is regulated by TREK‐1, a two‐pore‐domain (K2P) mechano‐gated potassium channel

Mechano‐gated two‐pore‐domain potassium (K2P) channels are expressed in the human bladder, with TREK‐1 being the predominant functional subunit. TREK‐1 channels in bladder smooth muscle are activated by membrane stretch and negative pressure applied to the patch pipette. Inhibition of TREK‐1 channels in the human detrusor significantly delays relaxation of bladder smooth muscle and triggers small‐amplitude spontaneous contractions in response to stretch. Application of negative pressure to cell‐attached patches (–20 mmHg) causes a 19‐fold increase in the open probability (NPo) of human TREK‐1 channels. l‐Methionine (1 mm) dramatically decreases the NPo of TREK‐1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Addition of arachidonic acid (10 μm) increases the open probability of methionine‐inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). TREK‐1 channels may serve as a promising pharmacological target for bladder dysfunction in humans.

Bladder outlet obstruction triggers neural plasticity in sensory pathways and contributes to impaired sensitivity in erectile dysfunction

Lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) are common problems in aging males worldwide. The objective of this work was to evaluate the effects of bladder neck nerve damage induced by partial bladder outlet obstruction (PBOO) on sensory innervation of the corpus cavernosum (CC) and CC smooth muscle (CCSM) using a rat model of PBOO induced by a partial ligation of the bladder neck. Retrograde labeling technique was used to label dorsal root ganglion (DRG) neurons that innervate the urinary bladder and CC. Contractility and relaxation of the CCSM was studied in vitro, and expression of nitric oxide synthase (NOS) was evaluated by Western blotting. Concentration of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide was measured by ELISA. Partial obstruction of the bladder neck caused a significant hypertrophy of the urinary bladders (2.5-fold increase at 2 wk). Analysis of L6-S2 DRG sections determined that sensory ganglia received input from both the urinary bladder and CC with 5-7% of all neurons double labeled from both organs. The contractile responses of CC muscle strips to KCl and phenylephrine were decreased after PBOO, followed by a reduced relaxation response to nitroprusside. A significant decrease in neuronal NOS expression, but not in endothelial NOS or protein kinase G (PKG-1), was detected in the CCSM of the obstructed animals. Additionally, PBOO caused some impairment to sensory nerves as evidenced by a fivefold downregulation of SP in the CC (P ≤ 0.001). Our results provide evidence that PBOO leads to the impairment of bladder neck afferent innervation followed by a decrease in CCSM relaxation, downregulation of nNOS expression, and reduced content of sensory neuropeptides in the CC smooth muscle. These results suggest that nerve damage in PBOO may contribute to LUTS-ED comorbidity and trigger secondary changes in the contraction/relaxation mechanisms of CCSM.

Estrous Cycle Dependent Fluctuations of Regulatory Neuropeptides in the Lower Urinary Tract of Female Rats upon Colon-Bladder Cross-Sensitization

Co-morbidity of bladder, bowel, and non-specific pelvic pain symptoms is highly prevalent in women. Little evidence is present on modulation of pelvic pain syndromes by sex hormones, therefore, the objective of this study was to clarify the effects of hormonal fluctuations within the estrous cycle on regulatory neuropeptides in female rats using a model of neurogenic bladder dysfunction. The estrous cycle in female rats (Sprague-Dawley, 230–250 g) was assessed by vaginal smears and weight of uterine horns. Neurogenic bladder dysfunction was induced by a single inflammatory insult to the distal colon. Protein expression of calcitonin gene related peptide (CGRP), substance P (SP), nerve growth factor (NGF), and brain derived neurotrophic factor (BDNF) in the pelvic organs, sensory ganglia and lumbosacral spinal cord was compared in rats in proestrus (high estrogen) vs diestrus (low estrogen). Under normal physiological conditions, concentration of SP and CGRP was similar in the distal colon and urinary bladder during all phases of the estrous cycle, however, acute colitis induced a significant up-regulation of CGRP content in the colon (by 63%) and urinary bladder (by 54%, p≤0.05 to control) of rats in proestrus. These changes were accompanied by a significant diminution of CGRP content in L6-S2 DRG after colonic treatment, likely associated with its release in the periphery. In rats with high estrogen at the time of testing (proestrus), experimental colitis caused a significant up-regulation of BDNF colonic content from 26.1±8.5 pg/ml to 83.4±32.5 pg/ml (N = 7, p≤0.05 to control) and also induced similar effects on BDNF in the urinary bladder which was also up-regulated by 5-fold in rats in proestrus (p≤0.05 to respective control). Our results demonstrate estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract upon colon-bladder cross-sensitization, which may contribute to pain fluctuations in female patients with neurogenic bladder pain.

Coronavirus-induced demyelination of neural pathways triggers neurogenic bladder overactivity in a mouse model of multiple sclerosis.

In the present study, we aimed to determine whether mice with coronavirus-induced encephalomyelitis (CIE) develop neurogenic bladder dysfunction that is comparable with the neurogenic detrusor overactivity observed in patients with multiple sclerosis. Adult mice (C57BL/6J, 8 wk of age, n = 146) were inoculated with a neurotropic strain of mouse hepatitis virus (A59 strain) and followed for 4 wk. Inoculation with the virus caused a significant neural deficit in mice with an average clinical symptom score of 2.6 ± 0.5 at 2 wk. These changes were accompanied by 25 ± 5% weight loss at 1 and 2 wk postinoculation (P ≤ 0.001 vs. baseline) followed by a recovery phase. Histological analysis of spinal cord sections revealed multifocal sites of demyelinated lesions. Assessment of micturition patterns by filter paper assay determined an increase in the number of small and large urine spots in CIE mice starting from the second week after inoculation. Cystometric recordings in unrestrained awake animals confirmed neurogenic bladder overactivity at 4 wk postinoculation. One week after inoculation with the A59 strain of mouse hepatitis virus, mice became increasingly sensitive to von Frey filament testing with responses enhanced by 45% (n = 8, P ≤ 0.05 vs. baseline at 4 g); however, this initial increase in sensitivity was followed by gradual and significant diminution of abdominal sensitivity to mechanical stimulation by 4 wk postinoculation. Our results provide direct evidence showing that coronavirus-induced demyelination of the central nervous system causes the development of a neurogenic bladder that is comparable with neurogenic detrusor overactivity observed in patients with multiple sclerosis.

Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

MP17-08 NEUROGENIC BLADDER DYSFUNCTION IN A MURINE MODEL OF MULTIPLE SCLEROSIS IS CAUSED BY CORONOVIRUS-INDUCED DEMYELINATION OF THE NERVOUS SYSTEM

INTRODUCTION AND OBJECTIVES: Neurogenic bladder dysfunction develops in patients with different neurodegenerative disorders including multiple sclerosis (MS). Lower urinary tract symptoms are reported in the majority of MS patients with the most common complaints of urinary urgency and frequency. The objective of this work was to evaluate neurogenic bladder dysfunction in a murine model of multiple sclerosis induced by a neurotrophic strain A59 of mouse hepatitis virus (MHV-A59). METHODS: Adult mice (C57BL/6J, 8 wks of age, N1⁄484) received single inoculation of MHV-A59 leading to the occurrence of coronavirus-induced encephalomyelitis (CIE). Animals were monitored daily for 4 wks for clinical signs of neurologic impairment. Clinical Symptom Score (CSS, 0-5) was assigned based on the level of tail tonicity, kyphosis and limb paresis/paralysis. Micturition patterns were assessed by filter paper assay and cystometric recordings in unrestrained mice. Contractile responses of the detrusor were evaluated in vitro by tension measurements in response to agonists and electric field stimulation (EFS). RESULTS: Inoculation with MHV-A59 virus induced a significant neural deficit with average CSS of 2.6 0.5 at 2 wks post-inoculation accompanied by 25 5 % of weight loss (p 0.001 to baseline). Histological analysis of spinal cord sections revealed multiple demyelinated lesions. Cystometric recordings confirmed neurogenic bladder overactivity at 4 wks post-inoculation including shortened inter-micturition interval, lower voided volume and elevated number of non-micturition contractions (p 0.05 to control group). In response to EFS, the cholinergic component of contraction was significantly reduced in mucosa intact CIE mouse bladders, while the atropine-resistant (purinergic) component was increased. Removal of the mucosa from CIE mouse bladders increased the cholinergic and decreased the purinergic components. CONCLUSIONS: Our results suggest that coronovirus-induced demyelination of the central nervous system causes the development of neurogenic bladder dysfunction that is similar to detrusor overactivity observed in MS patients. The underlying mechanisms may include an alteration in nerve-evoked contractions mediated by a urothelium dependent suppression of muscarinic and augmentation of purinergic mediated response in the bladder detrusor.

PD7-03 IMPAIRED EXPRESSION AND FUNCTION OF TREK-1, A STRETCH-ACTIVATED TWO-PORE DOMAIN POTASSIUM CHANNEL, UNDERLIES DETRUSOR OVERACTIVITY IN HUMANS

INTRODUCTION AND OBJECTIVES: Afferent signals during bladder filling and voiding are important in lower urinary tract (LUT) function. The sensory cation channel transient receptor potential channel vanilloid 4 (TRPV4) is a key mediator in the generation of afferent signals towards the central nervous system (CNS). It has become possible to study this central neural control by means of positron emission tomography (PET) of the brain. We hypothetized that deletion of TRPV4 would influence brain control of the LUT by decreasing afferent input. METHODS: 12 Female Sprague-Dawley rats and 10 trpv4-/rats were used. Under urethane anesthesia a catheter was inserted in the bladder and the urethra was ligated. The animal was placed in a FOCUS 220 PET scan. The catheter was connected to a syringe pump and pressure transducer. Wild type and trpv4-/rats were equally divided into two conditions; an empty bladder condition, without infusion, and a full bladder condition, with infusion of saline until spontaneous contractions occurred. During cystometry, brain imaging was performed after injection of [18F]-FDG in the tail vein. For analysis PET images were normalized to a stereotactic space based on the rat brain Paxinos atlas using PMOD . We analysed on voxel basis using SPM8. RESULTS: In WT rats clusters of increased FDG uptake in the full bladder condition were observed in the insular, cingulate and posterior temporal cortex, while uptake was decreased in a cerebellar cluster (fig). Trpv4-/showed a different activation pattern during bladder fullness, with increased uptake in the orbitofrontal cortex and cerebellar peduncle. A cluster of decreased FDG uptake was also detected in the cerebellum. Wild type and trpv4-/rats showed differences in glucose uptake in the cerebellar and temporal cortex, thalamus, hypothalamus and hippocampus. CONCLUSIONS: In wild type rats a full bladder activated the insular and cingulate cortex. Both regions are key areas in the central neural control of the bladder; the insular cortex being the main endpoint for bladder afferent signals and the cingulate cortex the origin of motivation to void. This brain activation pattern was not seen in trpv4-/rats representing decreased afferent input into the CNS that contributes to a diminished motivation to void. Source of Funding: none