Célia D. Cruz

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.

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, β-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.

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.

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.

Spinal Cord Injury and Bladder Dysfunction: New Ideas about an Old Problem

Control of the lower urinary tract (LUT) requires complex neuronal circuits that involve elements located at the peripheral nervous system and at different levels of the central nervous system. Spinal cord injury (SCI) interrupts these neuronal circuits and jeopardizes the voluntary control of bladder function. In most cases, SCI results in a period of bladder areflexia, followed by the emergence of neurogenic detrusor overactivity (NDO). Only recently, researchers have started to have a clearer vision ofthe mechanisms of SCI-induced changes affecting LUT control. For example, changes in the urothelium have recently been described and proposed to play a role in NDO. As such, a better understanding of NDO has generated new opportunities to investigate novel therapeutic approaches for NDO.In the present paper, we aim to update recent data concerning SCI-induced LUT dysfunction and therapeutic approaches commonly used to deal with NDO. We make a brief description of LUT control and changes occurring after SCI, and refer to new therapeutic options, including vanniloids and botulinum toxin. Finally, we discuss mechanisms of spinal cord repair, an interesting and very active area of investigation that has obtained some promising results in the recovery of LUT control.

Neurotrophins in Bladder Function: What Do We Know and Where Do We Go From Here?

Neurotrophins (NTs) have attracted considerable attention in the urologic community. The reason for this resides in the recognition of their ability to induce plastic changes of the neuronal circuits that govern bladder function. In many pathologic states, urinary symptoms, including urgency and urinary frequency, reflect abnormal activity of bladder sensory afferents that results from neuroplastic changes. Accordingly, in pathologies associated with increased sensory input, such as the overactive bladder syndrome (OAB) or bladder pain syndrome/interstitial cystitis (BPS/IC), significant amounts of NTs have been found in the bladder wall.

Sequestration of brain derived nerve factor by intravenous delivery of TrkB-Ig2 reduces bladder overactivity and noxious input in animals with chronic cystitis.

Brain derived nerve factor (BDNF) is a trophic factor belonging to the neurotrophin family. It is upregulated in various inflammatory conditions, where it may contribute to altered pain states. In cystitis, little is known about the relevance of BDNF in bladder-generated noxious input and bladder overactivity, a matter we investigated in the present study. Female rats were intraperitoneally (i.p.) injected with cyclophosphamide (CYP; 200 mg/kg). They received saline or TrkB-Ig(2) via intravenously (i.v.) or intravesical administration. Three days after CYP-injection, animals were anaesthetized and cystometries performed. All animals were perfusion-fixed and the spinal cord segments L6 collected, post-fixed and processed for c-Fos and phosphoERK immunoreactivity. BDNF expression in the bladder, as well as bladder histology, was also assessed. Intravesical TrkB-Ig(2) did not change bladder reflex activity of CYP-injected rats. In CYP-animals treated with i.v. TrkB-Ig(2) a decrease in the frequency of bladder reflex contractions, in comparison with saline-treated animals, was observed. In spinal sections from the latter group of animals, the number of phosphoERK and c-Fos immunoreactive neurons was lower than in sections from saline-treated CYP-animals. BDNF immunoreactivity was higher during cystitis but was not changed by TrkB-Ig(2) i.v. treatment. Evaluation of the bladder histology showed similar inflammatory signs in the bladders of inflamed animals, irrespective of the treatment. Data show that i.v. but not intravesical administration of TrkB-Ig(2) reduced bladder hyperactivity in animals with cystitis to levels comparable to those observed in unirritated rats. Since i.v. TrkB-Ig(2) also reduced spinal extracellular signal-regulated kinase (ERK) activation, it is possible that BDNF contribution to inflammation-induced bladder hyperactivity is via spinal activation of the ERK pathway. Finally, the reduction in c-Fos expression indicates that TrkB-Ig(2) also reduced bladder-generated noxious input. Our results show that sequestration of BDNF may be considered a new therapeutic strategy to treat chronic cystitis.

Transient receptor potential channels in bladder function

The transient receptor potential (TRP) superfamily of cationic ion channels includes proteins involved in the transduction of several physical and chemical stimuli to finely tune physiological functions. In the urinary bladder, they are highly expressed in, but not restricted to, primary afferent neurons. The urothelium and some interstitial cells also express several TRP channels. In this review, we describe the expression and the known roles of some members of TRP subfamilies, namely TRPV, TRPM and TRPA, in the urinary bladder. The therapeutic interest of modulating the activity of TRP channels to treat bladder dysfunctions is also discussed.

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.

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.