Mathieu Boudes

Functional characterization of a chronic cyclophosphamide‐induced overactive bladder model in mice

To describe a new mouse model of overactive bladder (OAB) at the histological level, pain, voiding behavior, and urodynamics, while assessing the physiological state of mice.

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.

Bladder dysfunction in a transgenic mouse model of multiple system atrophy.

Multiple system atrophy (MSA) is an adult‐onset neurodegenerative disorder presenting with motor impairment and autonomic dysfunction. Urological function is altered in the majority of MSA patients, and urological symptoms often precede the motor syndrome. To date, bladder function and structure have never been investigated in MSA models. We aimed to test bladder function in a transgenic MSA mouse featuring oligodendroglial α‐synucleinopathy and define its applicability as a preclinical model to study urological failure in MSA. Experiments were performed in proteolipid protein (PLP)–human α‐synuclein (hαSyn) transgenic and control wild‐type mice. Diuresis, urodynamics, and detrusor strip contractility were assessed to characterize the urological phenotype. Bladder morphology and neuropathology of the lumbosacral intermediolateral column and the pontine micturition center (PMC) were analyzed in young and aged mice. Urodynamic analysis revealed a less efficient and unstable bladder in MSA mice with increased voiding contraction amplitude, higher frequency of nonvoiding contractions, and increased postvoid residual volume. MSA mice bladder walls showed early detrusor hypertrophy and age‐related urothelium hypertrophy. Transgenic hαSyn expression was detected in Schwann cells ensheathing the local nerve fibers in the lamina propria and muscularis of MSA bladders. Early loss of parasympathetic outflow neurons and delayed degeneration of the PMC accompanied the urological deficits in MSA mice. PLP‐hαSyn mice recapitulate major urological symptoms of human MSA that may be linked to αSyn‐related central and peripheral neuropathology and can be further used as a preclinical model to decipher pathomechanisms of MSA.

Cav3.2 calcium channels: The key protagonist in the supraspinal effect of paracetamol

Summary Supraspinal Cav3.2 calcium channels are involved in analgesic effect of paracetamol through their inhibition following the activation of supraspinal TRPV1 receptors by AM404. ABSTRACT To exert its analgesic action, paracetamol requires complex metabolism to produce a brain‐specific lipoamino acid compound, AM404, which targets central transient receptor potential vanilloid receptors (TRPV1). Lipoamino acids are also known to induce analgesia through T‐type calcium‐channel inhibition (Cav3.2). In this study we show that the antinociceptive effect of paracetamol in mice is lost when supraspinal Cav3.2 channels are inhibited. Therefore, we hypothesized a relationship between supraspinal Cav3.2 and TRPV1, via AM404, which mediates the analgesic effect of paracetamol. AM404 is able to activate TRPV1 and weakly inhibits Cav3.2. Interestingly, activation of TRPV1 induces a strong inhibition of Cav3.2 current. Supporting this, intracerebroventricular administration of AM404 or capsaicin produces antinociception that is lost in Cav3.2−/− mice. Our study, for the first time, 1) provides a molecular mechanism for the supraspinal antinociceptive effect of paracetamol; 2) identifies the relationship between TRPV1 and the Cav3.2 channel; and 3) suggests supraspinal Cav3.2 inhibition as a potential pharmacological strategy to alleviate pain.

The use of cystometry in small rodents: a study of bladder chemosensation.

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladders nociceptive innervation and initiate voiding contractions that expel the bladders contents. Interestingly, the bladders sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion. After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions. Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Crucial Role of TRPC1 and TRPC4 in Cystitis-Induced Neuronal Sprouting and Bladder Overactivity

Purpose During cystitis, increased innervation of the bladder by sensory nerves may contribute to bladder overactivity and pain. The mechanisms whereby cystitis leads to hyperinnervation of the bladder are, however, poorly understood. Since TRP channels have been implicated in the guidance of growth cones and survival of neurons, we investigated their involvement in the increases in bladder innervation and bladder activity in rodent models of cystitis. Materials and Methods To induce bladder hyperactivity, we chronically injected cyclophosphamide in rats and mice. All experiments were performed a week later. We used quantitative transcriptional analysis and immunohistochemistry to determine TRP channel expression on retrolabelled bladder sensory neurons. To assess bladder function and referred hyperalgesia, urodynamic analysis, detrusor strip contractility and Von Frey filament experiments were done in wild type and knock-out mice. Results Repeated cyclophosphamide injections induce a specific increase in the expression of TRPC1 and TRPC4 in bladder-innervating sensory neurons and the sprouting of sensory fibers in the bladder mucosa. Interestingly, cyclophosphamide-treated Trpc1/c4−/− mice no longer exhibited increased bladder innervations, and, concomitantly, the development of bladder overactivity was diminished in these mice. We did not observe a difference neither in bladder contraction features of double knock-out animals nor in cyclophosphamide-induced referred pain behavior. Conclusions Collectively, our data suggest that TRPC1 and TRPC4 are involved in the sprouting of sensory neurons following bladder cystitis, which leads to overactive bladder disease.

Calcium-activated chloride current expression in axotomized sensory neurons: what for?

Calcium-activated chloride currents (CaCCs) are activated by an increase in intracellular calcium concentration. Peripheral nerve injury induces the expression of CaCCs in a subset of adult sensory neurons in primary culture including mechano- and proprioceptors, though not nociceptors. Functional screenings of potential candidate genes established that Best1 is a molecular determinant for CaCC expression among axotomized sensory neurons, while Tmem16a is acutely activated by inflammatory mediators in nociceptors. In nociceptors, such CaCCs are preferentially activated under receptor-induced calcium mobilization contributing to cell excitability and pain. In axotomized mechano- and proprioceptors, CaCC activation does not promote electrical activity and prevents firing, a finding consistent with electrical silencing for growth competence of adult sensory neurons. In favor of a role in the process of neurite growth, CaCC expression is temporally correlated to neurons displaying a regenerative mode of growth. This perspective focuses on the molecular identity and role of CaCC in axotomized sensory neurons and the future directions to decipher the cellular mechanisms regulating CaCC during neurite (re)growth.

Urodynamic changes in mice with experimental autoimmune encephalomyelitis correlate with neurological impairment.

Neurogenic bladder dysfunction is a major issue in Multiple Sclerosis (MS). High intravesical pressure should be treated early. Available therapies are insufficient and there is need for drug development and investigation of pathogenesis. Experimental Autoimmune Encephalomyelitis (EAE) in rodents is a well validated model to study MS. Previous research has shown that these animals develop urinary symptoms. However, from clinical studies, we know that symptoms do not necessarily reflect changes in bladder pressure. This paper aims to provide a complete overview of urodynamic changes in a model for detrusor overactivity in MS.

Urothelial TRPV1: TRPV1-Reporter Mice, a Way to Clarify the Debate?

The urothelium is a complex and dynamic epithelial layer with a sensory role contributing to mechano- and chemosensation in the bladder. This primary source of sensory inputs modulates micturition via an urothelial–sensory fibers crosstalk. The communication is mediated by ATP and NO release from epithelial cells. Therefore, a great interest has emerged in the urothelial molecular sensors during the last decade. Efforts were mainly focused on the transient receptor potential (TRP) channels. The TRP channels operate as polymodal cellular sensors involved in the fine tuning of many physiological processes. Based on sequence homology, the 28 mammalian members are divided into 6 families. TRPV1 and TRPV4 are the two most described members expressed in the urothelium. TRPV4 is a mechanosensitive channel involved in normal micturition (Gevaert et al., 2007) and has been highlighted as a putative pharmacological target to treat overactive bladder symptoms (Everaerts et al., 2010b). However, the extent to which functional TRPV1 channels are expressed in the bladder is debated. Experimental studies using TRPV1−/− mice suggested that TRPV1 might function as a mechanosensor in the bladder influencing the micturition threshold under general anesthesia (Birder et al., 2002). They also described urothelial ATP release upon capsaicin (the most potent TRPV1 agonist) application. The activation of TRPV1 by capsaicin induced intracellular calcium rise and inward cation current in both rat and human cultured urothelial cells (Charrua et al., 2009; Kullmann et al., 2009). The expression of TRPV1 in urothelium has been reported using diverse methods including quantitative PCR, immunohistochemistry, and western blot in different species (Lazzeri et al., 2004; Charrua et al., 2009; Heng et al., 2011). Recently, it has been shown that TRPV1 expression is not different from bladder dome and trigone in human biopsies at mRNA level (Sanchez Freire et al., 2011). In urothelial carcinoma, TRPV1 mRNA and protein levels were decreased (Lazzeri et al., 2005; Kalogris et al., 2010). The oral administration of a TRPV1 antagonist counteracted the bladder hyperactivity and the related hyperalgesia in cystitis animal model (Charrua et al., 2009). Altogether, these studies tend to demonstrate the functional expression of TRPV1 in urothelial cells and its implication in micturition in both physiological and pathological contexts. However, another school of thought exists. From that point of view, TRPV1 is expressed in small diameter bladder afferent fibers running through the urothelium but not by the epithelial cells themselves (Yamada et al., 2009; Yu et al., 2011). This expression is decreased following intradetrusor injections of botulinum toxin in patients (Apostolidis et al., 2005). Intravesical capsaicin and resiniferatoxin dissolved in high ethanol concentrations (5–30%) were able to suppress neurogenic detrusor overactivity in patients, but the relative role of the vanilloids and the ethanol have never been clarified (Ost et al., 2003). The specificity of TRPV1 antibodies have been questioned and appropriate controls (i.e., knock out animals) are not always used (Everaerts et al., 2009). All these studies question the TRPV1 expression in the urothelium. Moreover, a discrepancy also exists between the functional data. Indeed, two independent groups did not record TRPV1 positive signals (intracellular calcium rise or current) in cultured urothelial cells from guinea pig and mice (Xu et al., 2009; Everaerts et al., 2010a). The authors also doubted that Kullmann et al. (2009) recorded rat TRPV1 current considering that the current–voltage is linear whereas rat TRPV1 typical current is outwardly rectifying (Xu et al., 2005). However, TRPV1 gating properties are responsible for the rectification as TRPV1 single channel recordings are linear. In one hand, it is described that the stimulus strength can linearize the TRPV1 current–voltage relationship; therefore high concentration of capsaicin may modify the biophysical signature of the current. In the other hand, unknown adaptor proteins and/or subunits may exist in the urothelium; and this would be fascinating! In part, the lack of consensus reflects the limitations of traditional approaches to determine gene expression, including variable sensitivity, poor signal-to-noise, and lack of specificity. These divergent data need to be understood and explained to settle this important controversy for basic and clinical urology to determine whether TRPV1-based drugs could treat urothelial pathologies. Undoubtedly, researchers need new tools to assess this question. It might have been published in Journal of Neuroscience! In this study, the authors proposed to solve the mystery of TRPV1 expression in brain using a new genetic tool. They designed a TRPV1-reporter mouse using the insertion of two reporter genes after an IRES sequence. This genetic system allows the expression of a nuclear LacZ and the placental alkaline phosphatase (PLAP) with the putative TRPV1 expression pattern without disturbing TRPV1 function (Cavanaugh et al., 2011). These authors did not reveal any TRPV1 expression in bladder cDNA by PCR, but surprisingly they did not use their own reporter mice to confirm this result! They also created TRPV1 Cre mice that may help to distinguish differential expression and function among the urothelium layers via imaging and functional experiments. Nevertheless, scientists should bear in mind that gene expression under IRES sequence control is often inferior to the expression level of the upstream gene. Therefore, it could be possible that the reporter gene is not expressed or not detectable in case of low TRPV1 expression. However, these new transgenic mice might not be the panacea but they might surely help. These tools are now available, let us use them!

Cannabinoid receptor 1 also plays a role in healthy bladder

Cannabinoids are the active chemical component of Cannabis sativa (marijuana). The recreational and medical uses of cannabis go back over 5000 years. Cannabinoids produce a wide array of central and peripheral effects, some of which may have beneficial clinical applications. Indeed, cannabis extract-based medicines are used for the treatment of the nausea and vomiting associated with chemotherapy in patients with cancer and have been proposed to be useful in other neurological disorders because of their analgesic, antitumour, anti-inflammatory, and neuroprotective properties [1].