Central Control Circuit for Context-Dependent Micturition.

Urine release (micturition) serves an essential physiological function as well as a critical role in social communication in many animals. Here, we show a combined effect of olfaction and social hierarchy on micturition patterns in adult male mice, confirming the existence of a micturition control center that integrates pro- and anti-micturition cues. Furthermore, we demonstrate that a cluster of neurons expressing corticotropin-releasing hormone (Crh) in the pontine micturition center (PMC) is electrophysiologically distinct from their Crh-negative neighbors and sends glutamatergic projections to the spinal cord. The activity of PMC Crh-expressing neurons correlates with and is sufficient to drive bladder contraction, and when silenced impairs micturition behavior. These neurons receive convergent input from widespread higher brain areas that are capable of carrying diverse pro- and anti-micturition signals, and whose activity modulates hierarchy-dependent micturition. Taken together, our results indicate that PMC Crh-expressing neurons are likely the integration center for context-dependent micturition behavior.

PIEZO2 in sensory neurons and urothelial cells coordinates urination.

Henry Miller stated that "to relieve a full bladder is one of the great human joys". Urination is critically important in health and ailments of the lower urinary tract cause high pathological burden. Although there have been advances in understanding the central circuitry in the brain that facilitates urination1-3, there is a lack of in-depth mechanistic insight into the process. In addition to central control, micturition reflexes that govern urination are all initiated by peripheral mechanical stimuli such as bladder stretch and urethral flow4. The mechanotransduction molecules and cell types that function as the primary stretch and pressure detectors in the urinary tract mostly remain unknown. Here we identify expression of the mechanosensitive ion channel PIEZO2 in lower urinary tract tissues, where it is required for low-threshold bladder-stretch sensing and urethral micturition reflexes. We show that PIEZO2 acts as a sensor in both the bladder urothelium and innervating sensory neurons. Humans and mice lacking functional PIEZO2 have impaired bladder control, and humans lacking functional PIEZO2 report deficient bladder-filling sensation. This study identifies PIEZO2 as a key mechanosensor in urinary function. These findings set the foundation for future work to identify the interactions between urothelial cells and sensory neurons that control urination.

Model-based analysis of the acute effects of transcutaneous magnetic spinal cord stimulation on micturition after spinal cord injury in humans.

AIM: After spinal cord injuries (SCIs), patients may develop either detrusor-sphincter dyssynergia (DSD) or urinary incontinence, depending on the level of the spinal injury. DSD and incontinence reflect the loss of coordinated neural control among the detrusor muscle, which increases bladder pressure to facilitate urination, and urethral sphincters and pelvic floor muscles, which control the bladder outlet to restrict or permit bladder emptying. Transcutaneous magnetic stimulation (TMS) applied to the spinal cord after SCI reduced DSD and incontinence. We defined, within a mathematical model, the minimum neuronal elements necessary to replicate neurogenic dysfunction of the bladder after a SCI and incorporated into this model the minimum additional neurophysiological features sufficient to replicate the improvements in bladder function associated with lumbar TMS of the spine in patients with SCI. METHODS: We created a computational model of the neural circuit of micturition based on Hodgkin-Huxley equations that replicated normal bladder function. We added interneurons and increased network complexity to reproduce dysfunctional micturition after SCI, and we increased the density and complexity of interactions of both inhibitory and excitatory lumbar spinal interneurons responsive to TMS to provide a more diverse set of spinal responses to intrinsic and extrinsic activation of spinal interneurons that remains after SCI. RESULTS: The model reproduced the re-emergence of a spinal voiding reflex after SCI. When we investigated the effect of monophasic and biphasic TMS at two frequencies applied at or below T10, the model replicated the improved coordination between detrusor and external urethral sphincter activity that has been observed clinically: low-frequency TMS (1 Hz) within the model normalized control of voiding after SCI, whereas high-frequency TMS (30 Hz) enhanced urine storage. CONCLUSION: Neuroplasticity and increased complexity of interactions among lumbar interneurons, beyond what is necessary to simulate normal bladder function, must be present in order to replicate the effects of SCI on control of micturition, and both neuronal and network modifications of lumbar interneurons are essential to understand the mechanisms whereby TMS reduced bladder dysfunction after SCI.

A wireless closed-loop system for optogenetic peripheral neuromodulation.

The fast-growing field of bioelectronic medicine aims to develop engineered systems that can relieve clinical conditions by stimulating the peripheral nervous system1-5. This type of technology relies largely on electrical stimulation to provide neuromodulation of organ function or pain. One example is sacral nerve stimulation to treat overactive bladder, urinary incontinence and interstitial cystitis (also known as bladder pain syndrome)4,6,7. Conventional, continuous stimulation protocols, however, can cause discomfort and pain, particularly when treating symptoms that can be intermittent (for example, sudden urinary urgency)8. Direct physical coupling of electrodes to the nerve can lead to injury and inflammation9-11. Furthermore, typical therapeutic stimulators target large nerve bundles that innervate multiple structures, resulting in a lack of organ specificity. Here we introduce a miniaturized bio-optoelectronic implant that avoids these limitations by using (1) an optical stimulation interface that exploits microscale inorganic light-emitting diodes to activate opsins; (2) a soft, high-precision biophysical sensor system that allows continuous measurements of organ function; and (3) a control module and data analytics approach that enables coordinated, closed-loop operation of the system to eliminate pathological behaviours as they occur in real-time. In the example reported here, a soft strain gauge yields real-time information on bladder function in a rat model. Data algorithms identify pathological behaviour, and automated, closed-loop optogenetic neuromodulation of bladder sensory afferents normalizes bladder function. This all-optical scheme for neuromodulation offers chronic stability and the potential to stimulate specific cell types.

TRPV1 and mast cell involvement in repeated variate stress-induced urinary bladder dysfunction in adult female mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. Transient receptor potential vanilloid 1 (TRPV1) channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (P ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild-type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell-deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression, whereas control (no stress) mast cell-deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia (DRG) in WT mice exposed to RVS, but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.NEW & NOTEWORTHY Using pharmacological tools and transgenic mice in a repeated variate stress (RVS) model in female mice, we demonstrate that transient receptor potential vanilloid 1 (TRPV1) and mast cells contribute to the increased voiding frequency observed following RVS. TRPV1 and mast cells should continue to be considered as targets to improve bladder function in stress-induced bladder dysfunction.

Satellite Glial Cells Bridge Sensory Neuron Crosstalk in Visceral Pain and Cross-Organ Sensitization.

Following colonic inflammation, the uninjured bladder afferent neurons are also activated. The mechanisms and pathways underlying this sensory neuron cross-activation (from injured neurons to uninjured neurons) are not fully understood. Colonic and bladder afferent neurons reside in the same spinal segments and are separated by satellite glial cells (SGCs) and extracellular matrix in dorsal root ganglia (DRG). SGCs communicate with sensory neurons in a bidirectional fashion. This review summarizes the differentially regulated genes/proteins in the injured and uninjured DRG neurons and explores the role of SGCs in regulation of sensory neuron crosstalk in visceral cross-organ sensitization. The review also highlights the paracrine pathways in mediating neuron-SGC and SGC-neuron coupling with an emphasis on the neurotrophins and purinergic systems. Finally, I discuss the results from recent RNAseq profiling of SGCs to reveal useful molecular markers for characterization, functional study, and therapeutic targets of SGCs. SIGNIFICANCE STATEMENT: Satellite glial cells (SGCs) are the largest glial subtypes in sensory ganglia and play a critical role in mediating sensory neuron crosstalk, an underlying mechanism in colon-bladder cross-sensitization. Identification of novel and unique molecular markers of SGCs can advance the discovery of therapeutic targets in treatment of chronic pain including visceral pain comorbidity.

Nerve transfer for restoration of lower motor neuron-lesioned bladder, urethral and anal sphincter function. Part 4: Effectiveness of the motor reinnervation.

In pilot work, we showed that somatic nerve transfers can restore motor function in long-term decentralized dogs. We continue to explore the effectiveness of motor reinnervation in 30 female dogs. After anesthesia, 12 underwent bilateral transection of coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. Twelve months postdecentralization, eight underwent transfer of obturator nerve branches to pelvic nerve vesical branches, and sciatic nerve branches to pudendal nerves, followed by 10 mo recovery (ObNT-ScNT Reinn). The remaining four were euthanized 18 mo postdecentralization (Decentralized). Results were compared with 18 Controls. Squat-and-void postures were tracked during awake cystometry. None showed squat-and-void postures during the decentralization phase. Seven of eight ObNT-ScNT Reinn began showing such postures by 6 mo postreinnervation; one showed a return of defecation postures. Retrograde dyes were injected into the bladder and urethra 3 wk before euthanasia, at which point, roots and transferred nerves were electrically stimulated to evaluate motor function. Upon L2-L6 root stimulation, five of eight ObNT-ScNT Reinn showed elevated detrusor pressure and four showed elevated urethral pressure, compared with L7-S3 root stimulation. After stimulation of sciatic-to-pudendal transferred nerves, three of eight ObNT-ScNT Reinn showed elevated urethral pressure; all showed elevated anal sphincter pressure. Retrogradely labeled neurons were observed in L2-L6 ventral horns (in laminae VI, VIII, and IX) of ObNT-ScNT Reinn versus Controls in which labeled neurons were observed in L7-S3 ventral horns (in lamina VII). This data supports the use of nerve transfer techniques for the restoration of bladder function.NEW & NOTEWORTHY This data supports the use of nerve transfer techniques for the restoration of bladder function.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Denonvilliers' fascia as a potential nerve-course marker for the female urinary bladder.

OBJECTIVES: This study investigated the relationship between Denonvilliers' fascia (DF) and the pelvic plexus branches in women and explored the possibility of using the DF as a positional marker in nerve-sparing radical hysterectomy (RH). METHODS: This study included eight female cadavers. The DF, its lateral border, and the pelvic autonomic nerves running lateral to the DF were dissected and examined. The pelvis was cut into two along the mid-sagittal line. The uterine artery, deep uterine veins, vesical veins, and nerve branches to the pelvic organs were carefully dissected. RESULTS: The nerves ran sagitally, while the DF ran perpendicularly to them. The rectovaginal ligament was continuous with the DF, forming a single structure. The DF attached perpendicularly and seamlessly to the pelvic plexus. The pelvic plexus branches were classified into a ventral part branching to the bladder, uterus, and upper vagina and a dorsal part branching to the lower vagina and rectum as well as into four courses. Nerves were attached to the rectovaginal ligament and ran on its surface to the bladder ventral to the DF. The uterine branches split from the common trunk of these nerves. The most dorsal branch to the bladder primarily had a common trunk with the uterine branch, which is the most important and should be preserved in nerve-sparing Okabayashi RH. CONCLUSION: The DF can be used as a marker for nerve course, particularly in one of the bladder branches running directly superior to the DF, which can be preserved in nerve-sparing Okabayashi RH.

Detrusor nerve radiofrequency ablation (DENERA): Experimental study of safety and efficacy in an ovine model.

PURPOSE: The subtrigonal perivesical nerve plexus contains a large proportion of the bladder's innervation. A transurethral radiofrequency ablation approach has successfully denervated this region to alleviate overactive bladder symptoms, with some urothelial heat injury. We report a novel transvaginal RFA device (DENERA) and assess its feasibility and efficacy in denervating the perivesical nerve plexus of in vivo sheep. METHODS: In 14 adult female in vivo sheep, pulsed radiofrequency energy was applied transvaginally for three cycles of 4 min, maintaining the tissue temperature at 45°C, with 30 s of rest between each cycle. The control group (n = 4) was sacrificed without ablation, and various groups were sacrificed 1 week (n = 3), 4 weeks (n = 4), and 12 weeks (n = 3) after ablation. The bladder subtrigones were harvested then analyzed with H&E, S100, and TH immunostaining to quantify their neural density and neural vacuolization. RESULTS: The ablation procedure increased the neural vacuolization the most at 1 week and decreased the neural density the most at 4 weeks, with both variables displaying a significant change followed by a slight rebound towards baseline at 12 weeks. The H&E analysis showed that the needles penetrated deep into the subtrigonal detrusor muscle. The sheep recovered from the procedure with no complications or damage in the bladder wall or urothelium. CONCLUSIONS: This study shows that one DENERA treatment can cause subtrigonal denervation with some rebound afterwards and no complications. DENERA may become a promising OAB treatment option that can ablate the perivesical plexus without harming the urothelium.

Intraoperative pelvic neuromonitoring based on bioimpedance signals: a new method analyzed on 30 patients.

PURPOSE: Increasing importance has been attributed in recent years to the preservation of the pelvic autonomic nerves during rectal resection to achieve better functional results. In addition to improved surgical techniques, intraoperative neuromonitoring may be useful. METHODS: This single-arm prospective study included 30 patients who underwent rectal resection performed with intraoperative neuromonitoring by recording the change in the tissue impedance of the urinary bladder and rectum after stimulation of the pelvic autonomic nerves. The International Prostate Symptom Score, the post-void residual urine volume and the Low Anterior Resection Syndrome Score (LARS score) were assessed during the 12-month follow-up period. RESULTS: A stimulation-induced change in tissue impedance was observed in 28/30 patients (93.3%). In the presence of risk factors such as low anastomosis, neoadjuvant radiotherapy and a deviation stoma, an average increase of the LARS score by 9 points was observed 12 months after surgery (p = 0,04). The function of the urinary bladder remained unaffected in the first week (p = 0,7) as well as 12 months after the procedure (p = 0,93). CONCLUSION: The clinical feasibility of the new method for pelvic intraoperative neuromonitoring could be verified. The benefits of intraoperative pelvic neuromonitoring were particularly evident in difficult intraoperative situations with challenging visualization of the pelvic nerves.

Mechanism of Action of Tibial Nerve Stimulation in the Treatment of Lower Urinary Tract Dysfunction.

BACKGROUND AND OBJECTIVE: Tibial nerve stimulation (TNS) has long been used to effectively treat lower urinary tract dysfunction (LUTD). Although numerous studies have concentrated on TNS, its mechanism of action remains elusive. This review aimed to concentrate on the mechanism of action of TNS against LUTD. MATERIALS AND METHODS: A literature search was performed in PubMed on October 31, 2022. In this study, we introduced the application of TNS for LUTD, summarized different methods used in exploring the mechanism of TNS, and discussed the next direction to investigate the mechanism of TNS. RESULTS AND CONCLUSIONS: In this review, 97 studies, including clinical studies, animal experiments, and reviews, were used. TNS is an effective treatment for LUTD. The study of its mechanisms primarily concentrated on the central nervous system, tibial nerve pathway, receptors, and TNS frequency. More advanced equipment will be used in human experiments to investigate the central mechanism, and diverse animal experiments will be performed to explore the peripheral mechanism and parameters of TNS in the future.

Male Akita mice develop signs of bladder underactivity independent of NLRP3 as a result of a decrease in neurotransmitter release from efferent neurons.

Diabetic bladder dysfunction (DBD) is a prevalent diabetic complication that is recalcitrant to glucose control. Using the Akita mouse model (type 1) bred to be NLR family pyrin domain containing 3 (NLRP3)+/+ or NLRP3-/-, we have previously found that females (mild hyperglycemia) progress from an overactive to underactive bladder phenotype and that this progression was dependent on NLRP3-induced inflammation. Here, we examined DBD in the male Akita mouse (severe hyperglycemia) and found by urodynamics only a compensated underactive-like phenotype (increased void volume and decreased frequency but unchanged efficiency). Surprisingly, this phenotype was still present in the NLRP3-/- strain and so was not dependent on NLRP3 inflammasome-induced inflammation. To examine the cause of the compensated underactive-like phenotype, we assessed overall nerve bundle density and afferent nerve bundles (Aδ-fibers). Both were decreased in density during diabetes, but denervation was absent in the diabetic NLRP3-/- strain so it was deemed unlikely to cause the underactive-like symptoms. Changes in bladder smooth muscle contractility to cell depolarization and receptor activation were also not responsible as KCl (depolarizing agent), carbachol (muscarinic agonist), and α,ß-methylene-ATP (purinergic agonist) elicited equivalent contractions in denuded bladder strips in all groups. However, electrical field stimulation revealed a diabetes-induced decrease in contractility that was not blocked in the NLRP3-/- strain, suggesting that the bladder compensated underactive-like phenotype in the male Akita mouse is likely through a decrease in efferent neurotransmitter release.NEW & NOTEWORTHY In this study, we show that diabetic bladder dysfunction (the most common diabetic complication) manifests through different mechanisms that may be related to severity of hyperglycemia and/or sex. Male Akita mice, which have severe hyperglycemia, develop bladder underactivity as a result of a decrease in efferent neurotransmitter release that is independent of inflammation. This contrasts with females, who have milder hyperglycemia, where diabetic bladder dysfunction progresses from overactivity to underactivity in an inflammation-dependent manner.

Pathways and parameters of sacral neuromodulation in rats.

The stimulation paradigm for sacral neuromodulation has remained largely unchanged since its inception. We sought to determine, in rats, whether stimulation-induced increases in bladder capacity correlated with the proportion of sensory pudendal (PudS) neurons at each stimulated location (L6, S1). If supported, this finding could guide the choice of stimulation side (left/right) and level (S2, S3, S4) in humans. Unexpectedly, we observed that acute stimulation at clinically relevant (low) amplitudes [1-1.5 × motor threshold (Tm)], did not increase bladder capacity, regardless of stimulus location (L6 or S1). More importantly for the ability to test our hypothesis, there was little anatomic variation, and S1 infrequently contributed nerve fibers to the PudS nerve. During mapping studies we noticed that large increases in PudS nerve activation occurred at amplitudes exceeding 2Tm. Thus, additional cystometric studies were conducted, this time with stimulation of the L6-S1 trunk, to examine further the relationship between stimulation amplitude and cystometric parameters. Stimulation at 1Tm to 6Tm evoked increases in bladder capacity and decreases in voiding efficiency that mirrored those produced by PudS nerve stimulation. Many animal studies involving electrical stimulation of nerves of the lower urinary tract use stimulation amplitudes that exceed those used clinically (∼1Tm). Our results confirm that high amplitudes generate immediate changes in cystometric parameters; however, the relationship to low-amplitude chronic stimulation in humans remains unclear. Additional studies are needed to understand changes that occur with chronic stimulation, how these changes relate to therapeutic outcomes, and the contribution of specific nerve fibers to these changes.NEW & NOTEWORTHY Acute low-amplitude electrical stimulation of sacral nerve (sacral neuromodulation) did not increase bladder capacity in anesthetized CD, obese-prone, or obese-resistant rats. Increasing stimulation amplitude correlated with increases in bladder capacity and pudendal sensory nerve recruitment. It is unclear how the high-amplitude acute stimulation that is commonly used in animal experiments to generate immediate effects compares mechanistically to the chronic low-amplitude stimulation used clinically.

Imaging neuro-urodynamics of mouse major pelvic ganglion with a micro-endoscopic approach.

Postganglionic neurons of the autonomic nervous system lie outside of the central nervous system and innervate specific target effectors such as organs or glands. The major pelvic ganglion (MPG) is one such ganglion that plays a significant role in controlling bladder function in rodents. However, because of technical and physical constraints in recording electrophysiological signals from these neurons in vivo, the functional neural activity in MPG is mostly unknown. Transgenic animal models expressing genetically encoded calcium indicators now provide opportunities to monitor the activity of populations of neurons in vivo to overcome these challenges related to traditional electrophysiological methods. However, like many peripheral neurons, the MPG is not conducive to conventional fluorescent microscopy techniques, as it is located in the pelvic cavity, thus limiting robust optical access by benchtop microscopes. Here, we present an endoscopic approach based on a custom miniscope system (UCLA V3) that allows for effective in vivo monitoring of neural activity in the MPG for the first time. We show that our imaging approach can monitor activity of hundreds of MPG neurons simultaneously during the filling and emptying of the bladder in a urethane-anesthetized transgenic mouse line expressing GCaMP6s in cholinergic MPG neurons. By using custom analysis scripts, we isolated the activity of hundreds of individual neurons and show that populations of neurons have distinct phasic activation patterns during sequential bladder filling and voiding events. Our imaging approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.NEW & NOTEWORTHY The functional activity and information processing within autonomic ganglia is mostly unknown because of technical and physical constraints in recording electrophysiological signals from these neurons in vivo. Here, we use a micro-endoscopic approach to measure in vivo functional activity patterns from a population of autonomic neurons controlling bladder function for the first time. This approach can be adapted to record activity from autonomic neurons across different organs and systems in both healthy and disease models.

Efficacy of the magnetic stimulation of sacral nerve roots combined with Tui-na on neurogenic bladder after spinal cord injury: preliminary short-term results.

OBJECTIVE: Neurogenic bladder (NGB) is a serious complication after spinal cord injury (SCI), a destructive neurological disease. This study focused on exploring the efficacy of the magnetic stimulation of sacral nerve roots combined with Tui-na in treating NGB after SCI. METHODS: One hundred patients with NGB after SCI were studied, and intermittent clean catheterization was performed with a water intake program, and patients were grouped into four groups by the random number table method: general treatment group, Tui-na group, magnetic stimulation group, and combined treatment group. A series of relevant factors (voiding diary, urodynamics, and quality of life scores) and clinical efficacy of patients in the four groups before and after treatment were observed. RESULTS: The magnetic stimulation of sacral nerve roots alone, Tui-na alone, and the combination of both were all effective in improving bladder function and quality of life in patients with NGB after SCI, including improvements in voiding frequency, single urine output, maximum urine output, residual urine output, bladder volume and quality of life scores in patients with NGB after SCI. The efficacy of the magnetic stimulation of sacral nerve roots combined with Tui-na was better than that of the magnetic stimulation of sacral nerve roots alone and the Tui-na alone. CONCLUSION: This research demonstrates that the magnetic stimulation of sacral nerve roots combined with Tui-na treatment could effectively improve the urinary system and the quality of life of patients with NGB after SCI, which is worthy of clinical promotion and application.

Activation of MrgprA3 and MrgprC11 on Bladder-Innervating Afferents Induces Peripheral and Central Hypersensitivity to Bladder Distension.

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.

Spatiotemporal mapping of sensory and motor innervation of the embryonic and postnatal mouse urinary bladder.

The primary function of the urinary bladder is to store urine (continence) until a suitable time for voiding (micturition). These distinct processes are determined by the coordinated activation of sensory and motor components of the nervous system, which matures to enable voluntary control at the time of weaning. Our aim was to define the development and maturation of the nerve-organ interface of the mouse urinary bladder by mapping the organ and tissue distribution of major classes of autonomic (motor) and sensory axons. Innervation of the bladder was evident from E13 and progressed dorsoventrally. Increasing defasciculation of axon bundles to single axons within the muscle occurred through the prenatal period, and in several classes of axons underwent further maturation until P7. Urothelial innervation occurred more slowly than muscle innervation and showed a clear regional difference, from E18 the bladder neck having the highest density of urothelial nerves. These features of innervation were similar in males and females but varied in timing and tissue density between different axon classes. We also analysed the pelvic ganglion, the major source of motor axons that innervate the lower urinary tract and other pelvic organs. Cholinergic, nitrergic (subset of cholinergic) and noradrenergic neuronal cell bodies were present prior to visualization of these axon classes within the bladder. Examination of cholinergic structures within the pelvic ganglion indicated that connections from spinal preganglionic neurons to pelvic ganglion neurons were already present by E12, a time at which these autonomic ganglion neurons had not yet innervated the bladder. These putative preganglionic inputs increased in density prior to birth as axon terminal fields continued to expand within the bladder tissues. Our studies also revealed in numerous pelvic ganglion neurons an unexpected transient expression of calcitonin gene-related peptide, a peptide commonly used to visualise the peptidergic class of visceral sensory axons. Together, our outcomes enhance our understanding of neural regulatory elements in the lower urinary tract during development and provide a foundation for studies of plasticity and regenerative capacity in the adult system.

Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function.

Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.