Pelvic Ganglion Neurons Research Articles

An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

LIPUS-SCs-Exo promotes peripheral nerve regeneration in cavernous nerve crush injury-induced ED rats via PI3K/Akt/FoxO signaling pathway.

Clinical treatment of erectile dysfunction (ED) caused by cavernous nerve (CN) injury during pelvic surgery is difficult. Low-intensity pulsed ultrasound (LIPUS) can be a potential strategy for neurogenic ED (NED). However, whether Schwann cells (SCs) can respond to LIPUS stimulation signals is unclear. This study aims to elucidate the signal transmission between SCs paracrine exosome (Exo) and neurons stimulated by LIPUS, as well as to analyze the role and potential mechanisms of exosomes in CN repair after injury. The major pelvic ganglion (MPG) neurons and MPG/CN explants were stimulated with LIPUS of different energy intensities to explore the appropriate LIPUS energy intensity. The exosomes were isolated and purified from LIPUS-stimulated SCs (LIPUS-SCs-Exo) and non-stimulated SCs (SCs-Exo). The effects of LIPUS-SCs-Exo on neurite outgrowth, erectile function, and cavernous penis histology were identified in bilateral cavernous nerve crush injury (BCNI)-induced ED rats. LIPUS-SCs-Exo group can enhance the axon elongation of MPG/CN and MPG neurons compared to SCs-Exo group in vitro. Then, the LIPUS-SCs-Exo group showed a stronger ability to promote the injured CN regeneration and SCs proliferation compared to the SCs-Exo group in vivo. Furthermore, the LIPUS-SCs-Exo group increased the Max intracavernous pressure (ICP)/mean arterial pressure (MAP), lumen to parenchyma and smooth muscle to collagen ratios compared to the SCs-Exo group in vivo. Additionally, high-throughput sequencing combined with bioinformatics analysis revealed the differential expression of 1689 miRNAs between the SCs-Exo group and the LIPUS-SCs-Exo group. After LIPUS-SCs-Exo treatment, the phosphorylated levels of Phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and forkhead box O (FoxO) in MPG neurons increased significantly compared to negative control (NC) and SCs-Exo groups. Our study revealed that LIPUS stimulation could regulate the gene of MPG neurons by changing miRNAs derived from SCs-Exo, then activating the PI3K-Akt-FoxO signal pathway to enhance nerve regeneration and restore erectile function. This study had important theoretical and practical significance for improving the NED treatment.

Low-intensity pulsed ultrasound ameliorates erectile dysfunction induced by bilateral cavernous nerve injury through enhancing Schwann cell-mediated cavernous nerve regeneration.

Cavernous nerve injury-induced erectile dysfunction caused by pelvic surgery or trauma is refractory to conventional medications and required an alternative treatment. Low-intensity pulsed ultrasound is a noninvasive mechanical therapy that promotes nerve regeneration. To investigate the therapeutic effect and potential mechanism of low-intensity pulsed ultrasound in the treatment of neurogenic erectile dysfunction. Thirty rats were randomly divided into the sham-operated group, bilateral cavernous nerve injury group, and bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. The erectile function was assessed 3weeks after daily low-intensity pulsed ultrasound treatment. The penile tissues and cavernous nerve tissues were harvested and subjected to histologic analysis. Primary Schwann cells and explants were extracted from adult rats. The effects of low-intensity pulsed ultrasound on proliferation, migration, and nerve growth factor expression of Schwann cells and axonal elongation were examined in vitro. RNA sequencing and western blot assay were applied to predict and verify the molecular mechanism of low-intensity pulsed ultrasound-induced Schwann cell activation. Our study showed that low-intensity pulsed ultrasound promoted Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. Meanwhile, low-intensity pulsed ultrasound exhibits a stronger ability to enhance Schwann cells-mediated neurite outgrowth of major pelvic ganglion neurons and major pelvic ganglion/cavernous nerve explants in vitro. In vivo experiments demonstrated that the erectile function of the rats in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group was significantly higher than those in the bilateral cavernous nerve injury groups. Moreover, the expression levels of smooth muscle and cavernous endothelium also increased significantly in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group. In addition, we observed the higher density and number of cavernous nerve regenerating axons in the bilateral cavernous nerve injury + low-intensity pulsed ultrasound group, indicating that low-intensity pulsed ultrasound promotes axonal regeneration following cavernous nerve injury in vivo. RNA sequencing analysis and bioinformatic analysis suggested that low-intensity pulsed ultrasound might trigger the activation of the PI3K/Akt pathway. Western blot assay confirmed that low-intensity pulsed ultrasound activated Schwann cells through TrkB/Akt/CREB signaling. Low-intensity pulsed ultrasound promoted nerve regeneration and ameliorated erectile function by enhancing Schwann cells proliferation, migration, and neurotrophic factor nerve growth factor expression. The TrkB/Akt/CREB axis is the possible mechanism of low-intensity pulsed ultrasound-mediated Schwann cell activation. Low-intensity pulsed ultrasound-based therapy could be a novel potential treatment strategy for cavernous nerve injury-induced neurogenic erectile dysfunction.

Can Hybrid Synapse be Formed between Rat Spinal Motor Neurons and Major Pelvic Ganglion Neurons in vitro?

The purpose of this study is to determine whether synapses can be formed between spinal motor neurons (SMNs) and major pelvic ganglion (MPG) neurons of a rat in vitro. The green fluorescent protein (GFP)-labelled MPG cells were cultured together with SMNs in a specific medium. The synaptic-like contacts established between SMNs and MPG neurons were studied in co-cultures using morphologic and immunocytochemistry approaches. Phase-contrast observation of co-cultures showed apparent SMNs-MPG neurons contacts as early as three or four days in vitro. We demonstrate some evidence of synaptic contacts between SMNs and MPG neurons in vitro by immunostaining with antibody directed against postsynaptic density protein 95 (PSD-95). We describe the development process of a defined SMNs-MPG neurons co-culture system. The results suggest that the hybrid synapse formation that may occur between SMNs and MPG neurons in vitro played an essential role in the mechanisms of a regenerated bladder with an artificial somatic-autonomic reflex arc.

5-HT3 Signaling Alters Development of Sacral Neural Crest Derivatives That Innervate the Lower Urinary Tract.

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.

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.

Changes in the Neurochemical Coding of the Anterior Pelvic Ganglion Neurons Supplying the Male Pig Urinary Bladder Trigone after One-Sided Axotomy of Their Nerve Fibers.

The present study investigated the effect of unilateral axotomy of urinary bladder trigone (UBT)-projecting nerve fibers from the right anterior pelvic ganglion (APG) on changes in the chemical coding of their neuronal bodies. The study was performed using male pigs with immunohistochemistry and quantitative real-time PCR (qPCR). The animals were divided into a control (C), a morphological (MG) or a molecular biology group (MBG). APG neurons supplying UBT were revealed using the retrograde tracing technique with Fast Blue (FB). Unilateral axotomy resulted in an over 50% decrease in the number of FB+ neurons in both APG ganglia. Immunohistochemistry revealed significant changes in the chemical coding of FB+ cells only in the right ganglion: decreased expression of dopamine-B-hydroxylase (DBH)/tyrosine hydroxylase (TH) and up-regulation of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT), galanin (GAL), vasoactive intestinal polypeptide (VIP) and brain nitric oxide synthase (bNOS). The qPCR results partly corresponded with immunofluorescence findings. In the APGs, genes for VAChT and ChAT, TH and DBH, VIP, and NOS were distinctly down-regulated, while the expression of GAL was up-regulated. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.

SPARC: Building a Three‐Dimensional Atlas of Spinal Cord Circuits Regulating Lower Urinary Tract Function

Lumbar and sacral spinal cord circuits are critical for pelvic visceral regulation, as well as being engaged in pelvic pain states. Our study has focused on mapping two components of these circuits, the central projections of primary sensory neurons and the preganglionic neurons that drive motor activity via pelvic ganglia. These sensory pathways form a minority of lumbosacral dorsal root ganglion neurons. Likewise, although the parasympathetic preganglionic neurons are easily identified (aggregated in the sacral intermediolateral nucleus), the sympathetic preganglionic neurons comprise a minority of the thoracolumbar preganglionic neurons and are largely located outside of the intermediolateral nucleus. Our specific objectives were to map in adult male and female SD rats: (i) the terminal fields of primary sensory pathways that innervate distinct regions of the lower urinary tract; (ii) sympathetic and parasympathetic preganglionic neurons that innervate pelvic ganglion neurons, many of which regulate lower urinary tract tissues. To facilitate this, we have developed a spinal cord reference atlas, utilizing large volume immunostaining and clearing methods (iDISCO) to visualize primary neural classes throughout the entire lumbosacral cord. Region‐specific sensory neurons were identified by micro‐injection of CTB (B subunit of cholera toxin) into the bladder body, bladder trigone or urethra. In a separate group of animals, pelvic ganglion‐projecting preganglionic neurons were identified by microinjecting CTB bilaterally into the major pelvic ganglion (MPG). Four days later, spinal cord tissue was removed for immunohistochemical processing. We then combined several imaging approaches to reveal both mesoscopic and microscopic anatomical features of these pathways. Sensory terminals were mapped by constructing three‐dimensional representations, aligning alternate cryosections (TissueMaker, MicroBrightField) and relating CTB‐labelled terminals to specific chemoarchitectural features including spinal laminae and preganglionic neurons. Several molecular subclasses of sacral CTB‐labelled preganglionic neurons were mapped using the same strategy, while in other studies, the MPG‐projecting preganglionic neurons were mapped across the entire lumbosacral spinal cord, using immunohistochemistry of intact specimens and iDISCO clearing. Maps were constructed using 3D datasets obtained by light sheet microscopy or, for higher resolution analyses of neuronal connectivity and dendritic structure, ribbon scanning confocal microscopy. In conclusion, our tracing studies provide new insights into the complexity of sensory projections from defined pelvic visceral structures, and the location and structural properties of specific classes of pelvic viscera‐regulating preganglionic neurons. Our new spinal cord reference dataset provides a rich resource for mapping neural features following experimental intervention, for studies across the autonomic nervous system.Support or Funding InformationNIH SPARC 3OT2OD023872‐01S4

Spinal cord injury leads to atrophy in pelvic ganglia neurons

Among the most devastating sequelae of spinal cord injury (SCI) are genitourinary and gastrointestinal dysfunctions. Post-ganglionic neurons in pelvic ganglia (PG) directly innervate and regulate the function of the lower urinary tract (LUT), bowel, and sexual organs. A better understanding of how SCI affects PG neurons is essential to develop therapeutic strategies for devastating gastrointestinal and genitourinary complications ensuing after injury. To evaluate the impact of SCI on the morphology of PG neurons, we used a well- characterized rat model of upper thoracic SCI (T3 transection) that causes severe autonomic dysfunction. Using immunohistochemistry for neuronal markers, the neuronal profile size frequency distribution was quantified at one-, four-, and eight-weeks post SCI using recursive translation. Our investigation revealed an SCI-dependent leftward shift in neuronal size (i.e. atrophy), observable as early as one-week post injury. However, this effect was more pronounced at four and eight-weeks post-SCI. These findings demonstrate the first characterization of SCI-associated temporal changes in morphology of PG neurons and warrant further investigation to facilitate development of therapeutic strategies for recovery of autonomic functions following SCI.

Changes in excitability and ion channel expression in neurons of the major pelvic ganglion in female type II diabetic mice.

Bladder cystopathy and autonomic dysfunction are common complications of diabetes, and have been associated with changes in ganglionic transmission and some measures of neuronal excitability in male mice. To determine whether type II diabetes also impacts excitability of ganglionic neurons in females, we investigated neuronal excitability and firing properties, as well as underlying ion channel expression, in major pelvic ganglion (MPG) neurons in control, 10-week, and 21-week Leprdb/db mice. Type II diabetes in Leprdb/db animals caused a non-linear change in excitability and firing properties of MPG neurons. At 10 weeks, cells exhibited increased excitability as demonstrated by an increased likelihood of firing multiple spikes upon depolarization, decreased rebound spike latency, and overall narrower action potential half-widths as a result of increased depolarization and repolarization slopes. Conversely, at 21 weeks MPG neurons of Leprdb/db mice reversed these changes, with spiking patterns and action-potential properties largely returning to control levels. These changes are associated with numerous time-specific changes in calcium, sodium, and potassium channel subunit mRNA levels. However, Principal Components Analysis of channel expression patterns revealed that rectification of excitability is not simply a return to control levels, but rather a distinct ion channel expression profile in 21-week Leprdb/db neurons. These data indicate that type II diabetes can impact the excitability of post-ganglionic, autonomic neurons of female mice, and suggest that the non-linear progression of these properties with diabetes may be the result of compensatory changes in channel expression that act to rectify disrupted firing patterns of Leprdb/db MPG neurons.

Colonization of the Lower Urinary Tract by Neural Crest and Development of Pelvic Autonomic Innervation

The development of organs occurs concurrently with the formation of nerves that are essential for normal function. In the lower urinary tract, comprised of the bladder and urethra, appropriate innervation is essential for storage and coordinated release of urine at socially acceptable times. Understanding how the innervation of the lower urinary tract develops and the signaling pathways that regulate this process is likely to provide insight into congenital problems and offers a means to regenerate neural inputs in cases of damage or disease. The bladder and urethra receive both sensory and autonomic neural inputs that are derived from the neural crest during development. Sensory innervation of the bladder originates from neural crest‐derived dorsal root ganglia at thoracic and sacral spinal levels. Pelvic autonomic sympathetic and parasympathetic neurons that provide motor effects and play a principle role in coordinating bladder contractility are located within the discrete ganglia that lie near the neck of the bladder and proximal urethra. Formation of these mixed pelvic ganglia from neural crest, the routes of neural crest migration into the fetal urogenital sinus, timing of pelvic ganglia neural differentiation, and signaling pathways that regulate these processes are active areas of investigation that are being rapidly advanced through the use of mouse models. An overview of studies undertaken in transgenic mice to derive a spatiotemporal map of neural crest derivatives in the developing lower urinary tract as well as a timeline of differentiation of pelvic ganglia neurons and initial analysis of signaling pathways active during pelvic ganglia neuron differentiation will be presented.Support or Funding InformationThis work was supported by National Institutes of Health grants R01‐DK078158, RC1‐DK086594, and U01‐DK101038.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

980 - Major pelvic ganglion neurons express CXCR4, which binds stromal derived factor-1 and enhances neurotrophin protein levels and neurogenesis

980 - Major pelvic ganglion neurons express CXCR4, which binds stromal derived factor-1 and enhances neurotrophin protein levels and neurogenesis

017 Differential Effects on MPG Neurons and Penile Vasculature after Conformal Prostate Radiation in Rats

Prostatic radiation therapy (RT) is presumed to cause erectile dysfunction (ED) through damage to the vasculature and the nerves supplying the penis. This study examined the impact of prostatic RT on 1) vascular function in the internal pudendal artery (IPA) and penis and 2) the effect of in vivo prostate RT on the ex vivo survival and growth of major pelvic ganglia (MPG) neurons. Adult male Sprague-Dawley rats underwent conformal single fraction 25Gy RT using kilovoltage small animal microirradiator to the prostate or sham treatment. Erectile function was assessed by electrical stimulation of the cavernous nerve and ICP/MAP measurement at 2 or 10 weeks post-RT (n=10/grp). MPG were then excised; neurons were dissociated and cultured. Axon length and branching, and neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH) and TUNEL assay expression were measured in neuronal cultures at 72h. IPA and penises were excised and mounted in myograph tissue bath. Concentration response curves to phenylephrine (PE), acetylcholine (ACh), DEA NONOate, or sodium nitroprusside (SNP), and electrical field stimulated (EFS) contraction and NANC relaxation were assessed.

NLRP3/IL-1β mediates denervation during bladder outlet obstruction in rats.

Denervation of the bladder is a detrimental consequence of bladder outlet obstruction (BOO). We have previously shown that, during BOO, inflammation triggered by the NLRP3 inflammasome in the urothelia mediates physiological bladder dysfunction and downstream fibrosis in rats. The aim of this study was to assess the effect of NLRP3-mediated inflammation on bladder denervation during BOO. There were five groups of rats: (i) Control (no surgery); (ii) Sham-operated; (iii) BOO rats given vehicle; (iv) BOO rats given the NLRP3 inhibitor glyburide; and (v) BOO rats given the IL-1 receptor antagonist anakinra. BOO was constructed by ligating the urethra over a 1 mm catheter and removing the catheter. Medications were given prior to surgery and once daily for 12 days. Bladder sections were stained for PGP9.5, a pan-neuronal marker. Whole transverse sections were used to identify and count nerves while assessing cross-sectional area. For in vitro studies, pelvic ganglion neurons were isolated and treated with IL-1β. After a 48 h incubation apoptosis, neurite length and branching were assessed. In obstructed bladders, the number of nerves decreased while total area increased, indicating a loss of cell number and/or branching. The decrease in nerve density was blocked by glyburide or anakinra, clearly implicating the NLRP3 pathway in denervation. In vitro analysis demonstrated that IL-1β, a product of the inflammasome, induced apoptosis in pelvic ganglion neurons, suggesting one mechanism of BOO-induced denervation is NLRP3/IL-1β triggered apoptosis. The NLRP3/IL-1β-mediated inflammation pathway plays a significant role in denervation during BOO.

AB311. SPR-38 Clarification of the innervation of genitourinary structures: a neuronal tracing study in female mongrel hound dogs

ObjectiveMany studies focus on either afferent or efferent inputs (but not both), or on one structure of the genitourinary system. Only a few recent studies include information on inputs from sympathetic chain ganglia (SCG), and even fewer have examined the possibility of direct motor (autonomic or somatic) inputs from spinal cord ventral horns to genitourinary end organs. We sought to clarify origins of afferent and efferent information conveyed between the spinal cord, peripheral nervous system ganglia and genitourinary structures using retrograde and anterograde dye tracing methods.MethodsRetrograde dyes were injected into the bladder wall, EUS and clitoris of 14 female mongrel dogs (fluorogold, true blue, or nuclear yellow). Dorsal root ganglia (DRG), SCG, caudal mesenteric ganglion (CMG), pelvic plexus ganglia and spinal cord ventral horns were collected and examined for dye-labeled neuronal cell bodies. Detrusor muscle intramural ganglia were examined by injecting an anterograde dye (DiI) into the pelvic nerve’s anterior vesicle branch.ResultsRetrograde labeled cells were observed in several DRG, representative of afferent input from the bladder, EUS and clitoris. Anterograde labeling revealed a number of intramural ganglia in the bladder wall after distal pelvic nerve labeling. Sympathetic efferents included: (I) labeled cells in the CMG primarily from the bladder, yet small numbers from the EUS and clitoris; (II) labeled cells in SCG primarily from the bladder (widespread) and more localized input from EUS and clitoris; and (III) labeled cells in the intermediolateral cell column of thoracolumbar cord segments directly to the bladder and clitoris, a locale typically considered as sympathetic. Parasympathetic efferents included: (I) labeled neurons in pelvic plexus ganglia in bladder mesenteries; and (II) cells in lamina VII of sacral cord segments directly to the bladder and clitoris, a locale typically considered as sympathetic. Lastly, somatic (skeletal muscle) efferents to the EUS were evident as retrogradely labeled cells in sacral lamina IX cells.ConclusionsAfferent and efferent inputs to genitourinary structures are complex, yet a clear knowledge is needed to understand dysfunction after spinal cord injury and mechanisms underlying chronic pain syndromes in this region.Funding Source(s)NIH-NINDS NS070267

Pioglitazone Improves Pelvic Ganglion Neuronal Survival

Cavernosal nerve injury is a common complication after radical prostatectomy and causes erectile dysfunction (ED). Our recent publication established that pioglitazone (PGZ) improves cavernosal nerve function after crush injury in the rat model by both neural protection and neuroregeneration. This result is clinically significant for the many men who undergo treatment for localized prostate cancer. A better understanding of the effects of PGZ on pelvic ganglion neurons after cavernosal nerve injury is warranted. In this Research Highlight, we discuss the implications of our investigation from a molecular and clinical perspective.

Pioglitazone Enhances Survival and Regeneration of Pelvic Ganglion Neurons After Cavernosal Nerve Injury

To investigate the effects of pioglitazone on pelvic ganglion neurons in a rat model of bilateral cavernosal nerve crush injury (BCNI), thereby elucidating the actions of pioglitazone in preventing post-prostatectomy neurogenic erectile dysfunction. Sprague-Dawley rats aged 12 weeks were divided into four groups: (a) sham procedure, (b) BCNI, (c) BCNI + postsurgical pioglitazone, and (d) BCNI + pre and postsurgical pioglitazone (preventive therapy). Preoperative injection of Fluoro-Gold (FG) fluorescent tracer into the cavernosal tissue was performed for retrograde labeling of pelvic ganglion cells. Pelvic ganglia were resected at 2 weeks in all rats and processed for real-time polymerase chain reaction, immunohistochemistry, and Western blot to examine the expression of FG, neuronal nitric oxide synthase, β-III tubulin, neurturin, and glial cell line-derived neurotrophic factor family receptor alpha-2 (GFRα2). Animals treated with pre- and postsurgical pioglitazone demonstrated increased staining for FG similar to sham levels. Gene expression of neuronal nitric oxide synthase, neurturin, GFRα2, and β-III tubulin was also upregulated in the group receiving preventive therapy. Pioglitazone provides a protective effect on pelvic ganglion neurons after BCNI.

Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons

We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons.

Regeneration of sensory but not motor axons following visceral nerve injury

Following peripheral nerve injury, restoration of function may occur via the regeneration of injured axons or compensatory sprouting of spared axons. Injury to visceral nerves that control urogenital organs is a common consequence of pelvic surgery, however their capacity to reinnervate organs is poorly understood. To determine if and how sensory and motor connections to the bladder are re-established, a novel surgical model of visceral nerve injury was performed unilaterally in adult male Wistar rats. Bladder-projecting motor and sensory neurons in pelvic ganglia and lumbosacral dorsal root ganglia, respectively, were identified and characterised by retrograde tracing and immunofluorescence. Application of tracers ipsi- and contralateral to injury distinguished the projection pathways of new connections in the bladder. In naive animals, the majority of sensory and motor neurons project ipsilaterally to the bladder, while ~20 % project contralaterally and ~5 % bilaterally. Injured axons of motor neurons were unable to regenerate by 4weeks after transection. In contrast, by this time many injured sensory neurons regrew axons to reform a substantial plexus within the detrusor and suburothelial tissues. These regeneration responses were also indicated by upregulation of activating transcription factor-3 (ATF-3), which was sustained in motor neurons but transient in sensory bladder-projecting neurons. Axotomy had little or no effect on the survival of bladder-projecting sensory and motor neurons. We also found evidence that uninjured motor and sensory neurons develop additional projections to the denervated bladder tissue and return connectivity, likely by undergoing compensatory growth. In conclusion, our results show that visceral sensory and motor neurons have a different capacity to regenerate axons following axotomy, however in both components of the circuit uninjured bladder neurons spontaneously grow new axon collaterals to replace the lost terminal field within the organ. For a full functional recovery, understanding the environmental and cellular mechanisms that reduce the ability of pelvic ganglion cells to undergo axonal regeneration is needed.

Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons

Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and β4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO.