Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection.

Regeneration of adult mammalian central nervous system (CNS) axons is abortive, resulting in inability to recover function after CNS lesion, including spinal cord injury (SCI). Here, we show that the spiny mouse (Acomys) is an exception to other mammals, being capable of spontaneous and fast restoration of function after severe SCI, re-establishing hind limb coordination. Remarkably, Acomys assembles a scarless pro-regenerative tissue at the injury site, providing a unique structural continuity of the initial spinal cord geometry. The Acomys SCI site shows robust axon regeneration of multiple tracts, synapse formation, and electrophysiological signal propagation. Transcriptomic analysis of the spinal cord following transcriptome reconstruction revealed that Acomys rewires glycosylation biosynthetic pathways, culminating in a specific pro-regenerative proteoglycan signature at SCI site. Our work uncovers that a glycosylation switch is critical for axon regeneration after SCI and identifies ß3gnt7, a crucial enzyme of keratan sulfate biosynthesis, as an enhancer of axon growth.

TASCI-transcutaneous tibial nerve stimulation in patients with acute spinal cord injury to prevent neurogenic detrusor overactivity: protocol for a nationwide, randomised, sham-controlled, double-blind clinical trial.

INTRODUCTION: Neurogenic lower urinary tract dysfunction (NLUTD), including neurogenic detrusor overactivity (NDO) and detrusor sphincter dyssynergia, is one of the most frequent and devastating sequelae of spinal cord injury (SCI), as it can lead to urinary incontinence and secondary damage such as renal failure. Transcutaneous tibial nerve stimulation (TTNS) is a promising, non-invasive neuromodulatory intervention that may prevent the emergence of the C-fibre evoked bladder reflexes that are thought to cause NDO. This paper presents the protocol for TTNS in acute SCI (TASCI), which will evaluate the efficacy of TTNS in preventing NDO. Furthermore, TASCI will provide insight into the mechanisms underlying TTNS, and the course of NLUTD development after SCI. METHODS AND ANALYSIS: TASCI is a nationwide, randomised, sham-controlled, double-blind clinical trial, conducted at all four SCI centres in Switzerland. The longitudinal design includes a baseline assessment period 5-39 days after acute SCI and follow-up assessments occurring 3, 6 and 12 months after SCI. A planned 114 participants will be randomised into verum or sham TTNS groups (1:1 ratio), stratified on study centre and lower extremity motor score. TTNS is performed for 30 min/day, 5 days/week, for 6-9 weeks starting within 40 days after SCI. The primary outcome is the occurrence of NDO jeopardising the upper urinary tract at 1 year after SCI, assessed by urodynamic investigation. Secondary outcome measures assess bladder and bowel function and symptoms, sexual function, neurological structure and function, functional independence, quality of life, as well as changes in biomarkers in the urine, blood, stool and bladder tissue. Safety of TTNS is the tertiary outcome. ETHICS AND DISSEMINATION: TASCI is approved by the Swiss Ethics Committee for Northwest/Central Switzerland, the Swiss Ethics Committee Vaud and the Swiss Ethics Committee Zürich (#2019-00074). Findings will be disseminated through peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT03965299.

Chronic Pain After Spinal Cord Injury: Is There a Role for Neuron-Immune Dysregulation?

Spinal cord injury (SCI) is a devastating event with a tremendous impact in the life of the affected individual and family. Traumatic injuries related to motor vehicle accidents, falls, sports, and violence are the most common causes. The majority of spinal lesions is incomplete and occurs at cervical levels of the cord, causing a disruption of several ascending and descending neuronal pathways. Additionally, many patients develop chronic pain and describe it as burning, stabbing, shooting, or shocking and often arising with no stimulus. Less frequently, people with SCI also experience pain out of context with the stimulus (e.g., light touch). While abolishment of the endogenous descending inhibitory circuits is a recognized cause for chronic pain, an increasing number of studies suggest that uncontrolled release of pro- and anti-inflammatory mediators by neurons, glial, and immune cells is also important in the emergence and maintenance of SCI-induced chronic pain. This constitutes the topic of the present mini-review, which will focus on the importance of neuro-immune dysregulation for pain after SCI.

Underactive bladder in aging rats is associated with a reduced number of serotonin-expressing cells in the urethra and is improved by serotonin application to the urethra.

The aim of this study was to determine whether aging-related detrusor underactivity (DU) involves a decrease in 5-hydroxytryptamine (5-HT-positive)-expressing urethral cells and whether 5-HT stimulation of urethral sensory fibers improves detrusor function. Cystometries were performed in young (6 months) and aged (18-24 months) female Wistar rats. Aged rats with voiding contractions (VC) that were 2SD below the mean of those in young rats were considered to have DU. Bladder voiding efficiency (BVE) was calculated during saline or 5-HT solution cystometries. Rats were perfusion-fixed with a fixative solution (paraformaldehyde, PFA 4%) through the circulatory system and the urethra sectioned to count the number of 5-HT-immunoreactive (IR) cells. Isovolumetric cystometry was performed while irrigating the urethra with saline or 5µM-HT solution. Two-tailed unpaired t tests were used to determine the significance of differences. In aged DU rats, the mean (±SD) VC frequency was 0.24 ± 0.07 per minute, with an amplitude of 15 ± 3 cm H2 O. The mean (±SD) number of 5-HT-IR cells in the urethra of aged DU and young rats was 90 ± 11 and 182 ± 25, respectively (P < 0.01). 5-HT improved the mean (±SD) BVE of aged DU rats from 49 ± 3% to 78 ± 2% (P < 0.001). In isovolumetric cystometries, detrusor pressure during irrigation of the urethra with saline was 18 ± 1 cm H2 O, compared with 39 ± 2 cm H2 O during irrigation with 5-HT solution (P < 0.05). In rats, DU associated with aging is accompanied by a decrease in the number of 5-HT-positive cells. The results suggest that decreased 5-HT availability decreases urethral sensory fiber excitation, leading to a decrease the number of effective VC.

Urinary Neurotrophin Levels Increase in Women With Stress Urinary Incontinence After a Midurethral Sling Procedure.

OBJECTIVE: To investigate the association between urinary neurotrophin levels, maximum flow rate (Qmax) variation, and the appearance of urgency in women with stress urinary incontinence (SUI) after a midurethral sling (MUS) procedure. MATERIALS AND METHODS: Thirty-one women with SUI were treated with a MUS. One year later, the outcome of surgery and the onset of urgency were assessed. At baseline and 1-year follow-up, urine was collected to measure nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) concentration, and Qmax variation was calculated. Urine samples from healthy women (n = 20) without lower urinary tract symptoms and overactive bladder (OAB) wet patients (n = 32) were used as controls. Urinary neurotrophin levels were measured by enzyme-linked immunosorbent assay and normalized to creatinine concentration. RESULTS: At baseline, urinary levels of NGF and BDNF were similar between SUI and healthy women (NGF: 2.10 ± 0.68 vs 1.99 ± 1.05; BDNF: 1.99 ± 0.71 vs 1.81 ± 0.90), and significantly inferior to OAB wet patients (NGF: 2.10 ± 0.68 vs 2.50 ± 0.54, P < .05; BDNF: 1.99 ± 0.71 vs 2.71 ± 0.45, P < .05). After surgery, there was a significant increase of both neurotrophins (vs baseline, P < .05) to the values of OAB wet patients. Moreover, there was a significantly higher percentage increase of NGF in women with de novo urgency than in those without lower urinary tract symptoms (P = .019). A trend for a higher mean Qmax reduction in women with de novo urgency was also found (P = .085). CONCLUSION: These findings suggest that increased bladder outlet resistance after a MUS may play a key role in the rise of urinary neurotrophins, promoting sensitization of bladder primary afferents and causing de novo urgency in susceptible patients.

Co-administration of transient receptor potential vanilloid 4 (TRPV4) and TRPV1 antagonists potentiate the effect of each drug in a rat model of cystitis.

OBJECTIVE: To investigate transient receptor potential vanilloid 4 (TRPV4) expression in bladder afferents and study the effect of TRPV4 and TRPV1 antagonists, alone and in combination, in bladder hyperactivity and pain induced by cystitis. MATERIAL AND METHODS: TRPV4 expression in bladder afferents was analysed by immunohistochemistry in L6 dorsal root ganglia (DRG), labelled by fluorogold injected in the urinary bladder. TRPV4 and TRPV1 co-expression was also investigated in L6 DRG neurones of control rats and in rats with lipopolysaccharide (LPS)-induced cystitis. The effect of TRPV4 antagonist RN1734 and TRPV1 antagonist SB366791 on bladder hyperactivity and pain induced by cystitis was assessed by cystometry and visceral pain behaviour tests, respectively. RESULTS: TRPV4 is expressed in sensory neurones that innervate the urinary bladder. TRPV4-positive bladder afferents represent a different population than the TRPV1-expressing bladder afferents, as their co-localisation was minimal in control and inflamed rats. While low doses of RN1734 and SB366791 (176.7 ng/kg and 143.9 ng/kg, respectively) had no effect on bladder activity, the co-administration of the two totally reversed bladder hyperactivity induced by LPS. In these same doses, the antagonists partially reversed bladder pain behaviour induced by cystitis. CONCLUSIONS: TRPV4 and TRPV1 are present in different bladder afferent populations. The synergistic activity of antagonists for these receptors in very low doses may offer the opportunity to treat lower urinary tract symptoms while minimising the potential side-effects of each drug.

Can the adrenergic system be implicated in the pathophysiology of bladder pain syndrome/interstitial cystitis? A clinical and experimental study.

AIMS: To evaluate sympathetic system activity in bladder pain syndrome/interstitial cystitis (BPS/IC) patients and to investigate if chronic adrenergic stimulation in intact rats induces BPS/IC-like bladder modifications. METHODS: Clinical study--In BPS/IC patients and aged and body mass index matched volunteers TILT test was undertaken and catecholamines were measured in plasma and 24 hr urine samples. Experimental study--Phenylephrine was injected subcutaneously (14 days) to female Wistar rats. Pain behavior, spinal Fos expression, urinary spotting, number of fecal pellets expelled, frequency of reflex bladder contractions, and urothelial height were analyzed. Urothelium permeability was investigated by trypan blue staining. Immunoreactivity against caspase 3 and bax were studied in the urothelium and against alpha-1-adrenoreceptor and TRPV1 in suburothelial nerves. Mast cell number was determined in the sub-urothelium. In rats with lipopolysaccharide-induced cystitis, urinary catecholamines, and Vesicular Monoamine Transporter 2 (VMAT2) expression in bladder nerves were analyzed. RESULTS: The TILT test showed an increase of sympathetic activity. Noradrenaline levels in blood at resting conditions and in 24-hr urine samples were higher in BPS/IC patients. Phenylephrine administration increased visceral pain, spinal Fos expression, bladder reflex activity, urinary spotting and the number of expelled fecal pellets. The mucosa showed urothelial thinning and increased immunoreactivity for caspase 3 and bax. Trypan blue staining was only observed in phenylephrine treated animals. Suburothelial nerves co-expressed alpha1 and TRPV1. Mastocytosis was present in the suburothelium. Cystitis increased sympathetic nerve density and urinary noradrenaline levels. CONCLUSIONS: Excessive adrenergic stimulation of the bladder may contribute to the pathophysiological mechanisms of BPS/IC.

Biomarkers in lower urinary tract symptoms/overactive bladder: a critical overview.

PURPOSE OF REVIEW: Biomarkers constitute objectively measurable characteristics that can be evaluated as indicators of physiological and pathogenic processes and might be used as diagnostic, prognostic or predictive tools in clinical care. This review examines the availability of biomarkers to treat the dynamic and complex symptoms of overactive bladder (OAB). RECENT FINDINGS: OAB biomarkers may contribute to reveal the origin of storage symptoms in otherwise healthy individuals. The research encompassing the changes that occur in the bladder or in the peripheral (and central) nervous system might be determined through blood or urinary molecules (neurotrophins, ATP, prostaglandins, C-reactive protein and cytokines) or the measurement of events occurring in the bladder wall (bladder wall or detrusor wall thickness, oxyhemoglobin and deoxyhemoglobin concentration). These biomarkers might contribute to a better understanding of the pathophysiologic mechanisms underlying OAB. SUMMARY: The word biomarker to name all the parameters described above, from bladder wall thickness to urinary molecules, has been introduced to call the attention to a field wherein objective noninvasive parameters were nonexistent. OAB treatment based on a biomarker, in comparison to the treatment based on a diagnosis made from a careful history and exclusion of urinary tract infection, is not supported by current literature.

Urinary neurotrophic factors in healthy individuals and patients with overactive bladder.

PURPOSE: We investigated urinary levels of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in healthy individuals and patients with overactive bladder. MATERIALS AND METHODS: Urine from 40 healthy volunteers, half of them male and half female, was collected in the morning, afternoon and evening on 2 occasions 3 months apart. Morning urine samples were collected from 37 female naïve patients with overactive bladder. A total of 24 patients were followed. Urine was collected after a 3-month lifestyle intervention and after 3-month antimuscarinic treatment (oxybutynin 10 mg, extended release). Urinary nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor concentrations were measured by enzyme-linked immunosorbent assay and normalized to creatinine. Patients completed a 7-day bladder diary combined with an urgency severity scale. The number of urgency episodes per week was counted. RESULTS: In healthy individuals urinary levels of neurotrophic factors were stable. In patients with overactive bladder the nerve growth factor-to-creatinine (mean ± SD 488.5 ± 591.8 vs 188.3 ± 290.2, p = 0.005) and brain-derived neurotrophic factor-to-creatinine (mean 628.1 ± 590.5 vs 110.4 ± 159.5, p <0.001) ratios were significantly higher than in healthy women. No significant differences were found in the glial cell line-derived neurotrophic factor-to-creatinine ratio. After lifestyle intervention the nerve growth factor-to-creatinine and brain-derived neurotrophic factor-to-creatinine ratios decreased to a mean of 319.7 ± 332.3 and 432.5 ± 589.0 (vs baseline p = 0.318 and 0.033, respectively). After antimuscarinic treatment the nerve growth factor-to-creatinine and brain-derived neurotrophic factor-to-creatinine ratios further decreased to a mean of 179.8 ± 237.9 and 146.6 ± 264.9 (vs baseline p = 0.008 and <0.001, respectively). There was no significant variation in the glial cell line-derived neurotrophic factor-to-creatinine ratio at any time point. The reduction in the number of urgency episodes per week correlated with the brain-derived neurotrophic factor-to-creatinine variation (Pearson product-moment correlation coefficient r = 0.607, p = 0.006) but not with the nerve growth factor-to-creatinine ratio (r = 0.396, p = 0.094). CONCLUSIONS: The urinary nerve growth factor-to-creatinine and brain-derived neurotrophic factor-to-creatinine ratios are increased in patients with overactive bladder. These findings may have pathophysiological and clinical implications.

Neurotrophins as regulators of urinary bladder function.

Increased voiding frequency and urgency are among the most prevalent storage lower urinary tract symptoms (LUTS), often diagnosed as part of overactive bladder syndrome (OAB). It has been suggested that these symptoms are caused by excessive sensory activation of the neural micturition circuit. It seems likely that sensory pathway remodelling is also responsible for pain perception upon bladder filling in patients with bladder pain syndrome (BPS). Neurotrophins-including nerve growth factor (NGF), brain-derived nerve factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)-represent master modulators of neural plasticity, both in peripheral and central nervous systems. Accumulating evidence points towards a role for neurotrophins in the control of neural sensory function during micturition and indicates their involvement in the emergence of OAB-related and BPS-related LUTS. Neurotrophins could potentially be used as urinary biomarkers to improve diagnostic accuracy for OAB and BPS and monitor therapy effectiveness. Proof-of-principle clinical evidence has confirmed that NGF is a potential target for treating human bladder overactivity.

Transient receptor potential vanilloid 1 mediates nerve growth factor-induced bladder hyperactivity and noxious input.

OBJECTIVES: To explore the role of transient receptor potential vanilloid 1 (TRPV1) in the excitatory effects of chronic administration of nerve growth factor (NGF) on bladder-generated sensory input and reflex activity. To explore new therapeutic targets for bladder dysfunction. MATERIALS AND METHODS: Wild-type (WT) and TRPV1 knockout (KO) mice received daily intraperitoneal injections of NGF (1 µg/10 g) or saline for a period of 4 days, during which time thermal sensitivity was evaluated daily. On the 5th day, mice were anaesthetized and cystometries were performed. The frequency, amplitude and area under the curve (AUC) of bladder reflex contractions were determined. c-Fos expression was evaluated on L6 spinal cord sections of WT and TRPV1 KO mice treated with saline or chronic NGF by immunohistochemistry. TrkA receptor staining intensity was determined in L6 spinal cord sections and respective dorsal root ganglia of WT and TRPV1 KO mice. RESULTS: Repeated administration of NGF induced thermal hypersensitivity in WT but not in TRPV1 KO mice. The frequency of bladder contractions of saline-treated WT and TRPV1 KO mice was similar, the values respectively being 0.45 ± 0.12/min and 0.46 ± 0.16/min. Treatment with NGF enhanced bladder reflex activity in WT mice to 1.23 ± 0.41/min (P < 0.05). In NGF-treated KO mice, the frequency of bladder contractions was 0.60 ± 0.05/min. Irrespective of treatment, no differences were observed in the amplitude of bladder contractions of WT and TRPV1 KO mice. The AUC was significantly increased in NGF-treated WT-mice, when compared with saline-treated WT-mice. No changes were found in AUC of saline-treated and NGF-treated TRPV1 KO mice. Chronic administration of NGF resulted in a significant increase of spinal c-Fos expression in WT mice (P < 0.05 vs KO animals), but not in TRPV1 KO animals. TrkA expression was similar in WT and TRPV1 KO mice. CONCLUSIONS: NGF-induced bladder overactivity and noxious input depend on the interaction of NGF with TRPV1. The lack of bladder overactivity in TRPV1 KO mice treated with NGF does not represent loss of TrkA expression. TRPV1 is essential for NGF-driven bladder dysfunction and represents a bottleneck target in bladder pathologies associated with NGF up-regulation.

Spread of onabotulinumtoxinA after bladder injection. Experimental study using the distribution of cleaved SNAP-25 as the marker of the toxin action.

BACKGROUND: OnabotulinumtoxinA (Onabot/A) has been used to treat detrusor overactivity disorders. The treatment is based on several injections of toxin throughout the bladder wall. However, injection protocols are not well established among clinicians, varying in dose and dilution. OBJECTIVE: Study the distribution and neurochemistry of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) after Onabot/A administration in the guinea pig bladder. In addition, we analyzed which factor, dose or volume, contributes more to the diffusion of the toxin. DESIGN, SETTING, AND PARTICIPANTS: Guinea pig bladders were treated with Onabot/A via intramural injection or an instillation. MEASUREMENTS: Bladder cryostat sections were processed for single or dual immunohistochemistry staining with antibodies against cSNAP-25, vesicular acetylcholine transporter, tyrosine hydroxylase, and calcitonin gene-related peptide. Different administration methods and doses were analyzed. Statistical analysis was performed using the chi-square test for colocalization studies after multiple injections and the t test for the evaluation of affected fibers after a single injection. RESULTS AND LIMITATIONS: cSNAP-25 immunoreactive fibers were abundant throughout the bladder tissue in the mucosa and muscular layer. Double labeling showed that parasympathetic fibers are more affected than sympathetic or sensory. A single Onabot/A injection is more effective if diluted in a higher volume. Onabot/A instillation in the bladder does not cleave SNAP-25 protein. CONCLUSIONS: A single Onabot/A injection spreads the neurotoxin activity to the opposite side of the guinea pig bladder. This action is more evident when high saline volumes are used to dissolve Onabot/A. The toxin cleaves the SNAP-25 protein mainly in cholinergic but also in adrenergic and sensory fibers. In contrast with intramural injection, instillation of Onabot/A does not cleave SNAP-25 in nerve fibers.

Effect of onabotulinumtoxinA on intramural parasympathetic ganglia: an experimental study in the guinea pig bladder.

PURPOSE: We investigated whether onabotulinumtoxinA injected in the bladder would affect preganglionic parasympathetic nerve endings in intramural ganglia. MATERIALS AND METHODS: Guinea pig bladders were injected with 5 U of botulinum toxin. At 24 hours bladders were collected and processed for immunohistochemistry using tyrosine hydroxylase, and intact and cleaved SNAP-25. To identify the different populations of affected fibers coursing the ganglia we performed double immunoreactions for cleaved SNAP-25 and VAChT, TH or CGRP. RESULTS: VAChT immunoreactive fibers were identified in axons and varicosities of presynaptic to postganglionic parasympathetic neurons. Those fibers were also immunoreactive to SV2 and SNAP-25. The rare CGRP and TH immunoreactive fibers coursing in the ganglia did not express SV2 or SNAP-25. After onabotulinumtoxinA injection the cleaved form of SNAP-25 was abundantly expressed in parasympathetic fibers. CONCLUSIONS: Botulinum toxin injection in the bladder wall affects preganglionic parasympathetic nerve terminals. This could contribute to the strong effect of botulinum toxin on bladder smooth muscle activity.

Rat detrusor overactivity induced by chronic spinalization can be abolished by a transient receptor potential vanilloid 1 (TRPV1) antagonist.

PURPOSE: To evaluate the effect of a transient receptor potential vanilloid 1 (TRPV1) antagonist GRC 6211 on neurogenic detrusor overactivity (NDO) of spinal origin. MATERIALS AND METHODS: Cystometries under urethane anaesthesia were obtained in 14 chronic spinalized rats to confirm NDO. Two groups were created. In the first one (n=10), GRC 6211 (0.01, 0.1 and 1mg/kg weight) was administered via the duodenum in cumulative doses and cystometries performed 150 min after the administration of each dose of the drug. In the second group (n=4), used as control, the animals were submitted to cystometries during 12 hours, without administration of GRC 6211. Frequency and amplitude of bladder contractions were recorded in both groups. RESULTS: The mean (±SDev) bladder detrusor muscle contraction frequency of spinalized rats was 0.7±0.27 contractions/min. GRC 6211 produced a significant dose-dependent effect, with the frequency diminished to 0.53±0.23, 0.40±0.20 and 0.20±0.13 contractions/min, respectively. The mean (± SDev) amplitude of bladder contractions was 48.4±4.4 cmH(2)O. After administration of 0.01 mg/kg, 0.1mg/kg and 1mg/kg of GRC 6211, the amplitude decreased to 47.1±4.3, 45.6±5.6 and 40.2±4.1 cm H(2)O respectively. The effect was significant at 0.1 and 1mg/kg doses. Cystometries performed in the control group of spinalized rats showed no evidence of detrusor fatigue caused by the urethane anaesthesia and long duration of the experiment. CONCLUSION: TRPV1 antagonists may be very effective in reducing NDO of spinal origin. This finding may have profound implications for the pathogenesis and future treatment options of patients with spinal NDO.

Nerve growth factor in bladder dysfunction: contributing factor, biomarker, and therapeutic target.

In the last two decades, nerve growth factor (NGF), initially described as a prototypical trophic factor in the development of sensory and sympathetic innervation, has emerged as a complex regulator of neural plasticity along the micturition pathways. This review aims to summarize the current experimental and clinical evidence for a role of NGF in urinary bladder. Experimental administration of NGF elicits the states of increased sensation, urgency, and bladder hyperreflexia, resembling pathologies associated with bladder overactivity and inflammatory pain, such as overactive bladder syndrome (OAB) and interstitial cystitis/painful bladder syndrome (IC/PBS). There is strong experimental evidence, including the effective therapeutic targeting, on the direct causal role of NGF in rodent models of bladder outlet obstruction, spinal cord injury, diabetic bladder dysfunction, and interstitial inflammation. In humans, there are attempts to employ urinary NGF levels as a diagnostic marker in various forms of OAB and IC/PBS. In near future, use of novel experimental tools, such as urothelium-specific NGF transgenic mice or more specific low-molecular weight NGF receptor modulators, may provide better understanding of several unresolved issues in NGF-related bladder dysfunction. Moreover, successful experimental therapeutic approaches, such as NGF sequestering proteins or modified NGF antibodies, await the translation to the clinical treatment of bladder disorders.

Distribution of the high-affinity binding site and intracellular target of botulinum toxin type A in the human bladder.

BACKGROUND: Botulinum toxin type A (BoNTA) has been successfully used in the treatment of refractory detrusor overactivity. The toxin is internalized after binding a high-affinity receptor, synaptic vesicle protein 2 (SV2), which is exposed in the cell membrane during the exocytosis process. In the cytoplasm, BoNTA cleaves specific sites of synaptosomal-associated protein 25 (SNAP-25), preventing the assembly of the synaptic fusion complex SNARE and blocking exocytosis. OBJECTIVE: In the present work, the distribution of SV2 and SNAP-25 was first investigated in human bladders. The neurochemistry of BoNTA-sensitive structures was then investigated using markers for parasympathetic, sympathetic, and sensory fibers. DESIGN, SETTING, AND PARTICIPANTS: Human bladders were obtained from cadaveric organ donors (age range: 19-74 yr). MEASUREMENTS: Bladder sections were processed for single or dual immunofluorescence staining with antibodies against SV2, SNAP-25, ß-3 tubulin, vesicular acetylcholine transporter, tyrosine hydroxilase, and calcitonin gene-related peptide. RESULTS AND LIMITATIONS: SV2 and SNAP-25 immunoreactive fibers were distributed throughout the suburothelium and muscular layer. Double labeling showed extensive colocalization of both proteins in nerve fibers. SV2 is more expressed in parasympathetic fibers than in sympathetic or sensory fibers. No expression was found in urothelial or muscular cells. Because only normal bladders were used, this distribution should be applied with caution to pathologic bladders. CONCLUSIONS: SV2 and SNAP-25 colocalize abundantly throughout the urinary bladder. SV2 is more abundant in cholinergic, parasympathetic fibers. These nerves are suggested to be the main target for BoNTA action in the human urinary bladder.

GRC-6211, a new oral specific TRPV1 antagonist, decreases bladder overactivity and noxious bladder input in cystitis animal models.

PURPOSE: We evaluated the effects of GRC-6211, an orally active TRPV1 antagonist, on the function and noxious input of naïve and inflamed bladders. MATERIALS AND METHODS: In urethane (Sigma(R)) anesthetized rats 0.5 ml GRC-6211 (0.001, 0.01, 0.1 and 1 mg/kg weight) or its vehicle (0.5% methylcellulose) were administered through a duodenal catheter and cystometry was done during infusion of saline, 100 microM capsaicin or 0.5% acetic acid (Merck, Feltham, United Kingdom). Cystometry was also performed in WT and TRPV1 knockout mice treated with 1 mg/kg GRC-6211. Cystometry was done in rats inflamed with lipopolysaccharide after receiving 0.1 mg/kg GRC-6221 or vehicle. Spinal c-fos expression induced by 0.5% acetic acid was investigated after 0.1 mg/kg GRC-6211 or vehicle administration. TRPV1 immunoreactivity was evaluated in the bladder after GRC-6211 administration. RESULTS: The reflex activity of rat and WT mice naïve bladders was unchanged by GRC-6211 up to a dose of 0.1 mg/kg. At 1 mg/kg contractions were transiently suppressed in naïve rats and WT mice but not in TRPV1 knockout mice. GRC-6211 (0.1 mg/kg) completely prevented capsaicin induced irritation, while the 0.001, 0.01 or 0.1 mg/kg dose decreased the mean +/- SD frequency of bladder contractions during acetic acid infusion from 1.5 +/- 0.3 to 1.35 +/- 0.35 (not significant), 0.9 +/- 0.2 (p <0.05) and 0.8 +/- 0.2 (p <0.05), respectively. Lipopolysaccharide inflamed rats had 1.4 +/- 0.4 and 0.8 +/- 0.1 contractions per minute after vehicle and GRC-6211, respectively (p <0.05). The c-fos expression induced by acetic acid was decreased by GRC-6211 (85.5 +/- 19.1 to 46.7 +/- 9.4, p <0.05). GRC-6211 did not change bladder TRPV1 immunoreactivity. CONCLUSIONS: GRC-6211 counteracts the bladder hyperactivity and noxious input induced by cystitis. At high doses it suppresses normal bladder activity by a TRPV1 dependent mechanism. TRPV1 antagonists might be useful for cystitis.

Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals.

Recently, it has been demonstrated that intrathecal delivery of resiniferatoxin (RTX) produces strong analgesia, even in models of bone cancer pain. RTX has been investigated to treat bladder dysfunction of spinal origin, applied by intravesical instillation. However, RTX delivered by this route was not completely satisfactory in controlling urinary incontinence and high intravesical pressure. Thus, the present study assessed the effects of intrathecal injections of RTX in bladder dysfunction in rats with spinal cord transection (SCT). Bladder function was evaluated in SCT rats 24 h following intrathecal administration of RTX. Detrusor overactivity and intravesical pressure were reduced in a dose-dependent manner. This was accompanied by a decrease in spinal cord TRPV1 and CGRP, but not in IB4 binding sensory fibres. Also, intrathecal RTX induced a dose-dependent reduction in spinal cord activation of the ERK pathway. Overall, our results show that intrathecal administration of RTX effectively reduces detrusor overactivity and reduces intravesical pressure in models of complete chronic spinal cord transection by suppressing the activity of TRPV1 expressing afferent fibres. Also, intrathecal RTX decreases sensory input, as shown by reduced spinal ERK activation. These findings might be relevant for the management of patients with spinal cord injuries.

The activation of the ERK pathway contributes to the spinal c-fos expression observed after noxious bladder stimulation.

C-fos is an immediate-early gene whose expression in the spinal cord has been extensively used as a marker of peripheral noxious stimulation. The Fos protein accumulates in the nuclei of spinal neurons, reaching detectable levels 2 h after stimulation. The ERK pathway is an important signalling pathway in spinal cord neurons. ERK is activated upon phosphorylation on specific amino acid residues. Its activation in the spinal cord, following noxious stimulation, has been shown to contribute to the establishment and maintenance of long-term neuronal alterations associated with chronic pain. Phosphorylated ERK can target several cellular elements, including transcription factors, which indicates that ERK participates in the regulation of gene expression. The relation between ERK and c-fos is at present still unclear. Some in vitro studies have reached the conclusion that ERK contributes to c-fos regulation whereas others have provided evidence of ERK-independent c-fos expression. In fact, in the spinal cord the occurrence of c-fos expression in the absence of ERK phosphorylation has been reported. In this study we investigated in vivo the contribution of ERK to c-fos expression in the spinal cord. By inhibiting spinal ERK activation with intrathecal administration of PD98059, we verified that ERK phosphorylation does contribute to regulate c-fos expression upon noxious bladder stimulation.