Pelvic Ganglion Research Articles

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

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.

Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model

IntroductionThe human placenta provides a bountiful and noncontroversial source of stem cells which have the potential for regeneration of injured tissue. These cells may restore erectile function after neurovascular tissue injury such as that seen in radical pelvic surgeries and pelvic trauma. AimTo determine the effect of human placenta–derived stem cells on erectile function recovery and histological changes at various time points in a cavernous nerve injury rat model and to study the fate of injected stem cells throughout the regenerative process. MethodsHuman placental stem cells (PSCs) were dual labeled with monomeric Katushka far red fluorescent protein (mKATE)-renLUC using a lentivirus vector. A pelvic neurovascular injury–induced erectile dysfunction model was established in male, athymic rats by crushing the cavernous nerves and ligating the internal pudendal neurovascular bundles, bilaterally. At the time of defect creation, nonlabeled PSCs were injected into the corpus cavernosum at a concentration of 2.5 × 106 cells/0.2 mL. The phosphate-buffered saline–treated group served as the negative control group, and age-matched rats (age-matched controls) were used as the control group. Erectile function, histomorphological analyses, and Western blot were assessed at 1, 6, and 12 weeks after model creation. The distribution of implanted, dual-labeled PSCs was monitored using an in vivo imaging system (IVIS). Implanted cells were further tracked by detection of mKATE fluorescence in histological sections. Main Outcome MeasureThe main outcome measure includes intracavernous pressure/mean arterial pressure ratio, neural, endothelial, smooth muscle cell regeneration, mKATE fluorescence, and IVIS imaging. ResultsThe ratio of intracavernous pressure to mean arterial pressure significantly increased in PSC-injected rats compared with phosphate-buffered saline controls (P < 0.05) at the 6- and 12-week time points, reaching 72% and 68% of the age-matched control group, respectively. Immunofluorescence staining and Western blot analysis showed significant increases in markers of neurons (84.3%), endothelial cells (70.2%), and smooth muscle cells (70.3%) by 6 weeks in treatment groups compared with negative controls. These results were maintained through 12 weeks. IVIS analysis showed luminescence of implanted PSCs in the injected corpora immediately after injection and migration of cells to the sites of injury, including the incision site and periprostatic vasculature by day 1. mKATE fluorescence data revealed the presence of PSCs in the penile corpora and major pelvic ganglion at 1 and 3 days postoperatively. At 7 days, immunofluorescence of penile PSCs had disappeared and was diminished in the major pelvic ganglion. Clinical ImplicationsPlacenta-derived stem cells may represent a future “off-the-shelf” treatment to mitigate against development of erectile dysfunction after radical prostatectomy or other forms of pelvic injury. Strength & LimitationsSingle dose injection of PSCs after injury resulted in maximal functional recovery and tissue regeneration at 6 weeks, and the results were maintained through 12 weeks. Strategies to optimize adult stem cell therapy might achieve more effective outcomes for human clinical trials. ConclusionHuman PSC therapy effectively restores the erectile tissue and function in this animal model. Thus, PSC therapy may provide an attractive modality to lessen the incidence of erectile dysfunction after pelvic neurovascular injury. Further improvement in tissue regeneration and functional recovery may be possible using multiple injections or systemic introduction of stem cells.Gu X, Thakker PU, Matz EL, et al. Dynamic Changes in Erectile Function and Histological Architecture After Intracorporal Injection of Human Placental Stem Cells in a Pelvic Neurovascular Injury Rat Model. J Sex Med 2020;17:400–411.

Co-overexpression of VEGF and GDNF in adipose-derived stem cells optimizes therapeutic effect in neurogenic erectile dysfunction model.

ObjectivesTo evaluate the rapid repair potential of adipose‐derived stem cells (ADSCs) co‐overexpressing VEGF and GDNF on bilateral cavernous nerve injury (BCNI) in rat models. Progressive fibrosis of the penis that occurs shortly after BCNI is a key cause of clinical treatment difficulty of erectile dysfunction (ED). Traditional medications are ineffective for ED caused by BCNI. ADSCs have shown therapeutic effects in animal models, but disappointing in clinical treatment suggests that we should explore optimal treatment of it.Materials and methodsWe extracted ADSCs from rat epididymis. Lentiviral transfection was verified by western blot and immunofluorescence. Thirty‐six SD rats (10 weeks old) were randomly divided into six groups (n = 6 per group): sham surgery, and remaining five BCNI groups transplanted PBS or ADSCs which were genetically modified by vehicle, VEGF (ADSC‐V), GDNF (ADSC‐G), or VEGF&GDNF (ADSC‐G&V) around major pelvic ganglion (MPG). We investigated the therapeutic effects of BCNI rat model which is characterized by ED, penile tissue fibrosis and hypoxia, and lack of nitrogen nerves or vascular atrophy.ResultsErectile function was almost recovered after 2 weeks of transplantation of ADSC‐G&V, promoted cavernous nerve repair, prevented penile fibrosis and preserving the vascular endothelium, which was significant differences amongst ADSC‐V or ADSC‐G. Moreover, GM‐ADSCs were detected in MPG and penis, indicating that their participation in repair of target organs and transverse nerves.ConclusionsThese promising data indicate that ADSCs co‐overexpressed VEGF and GDNF‐induced synergistic effects, make it a potential tool for recovering of erectile function speedily after BCNI.

Effect of castration on pelvic neurons in the male pig.

The present study investigated the influence of castration performed at neonatal age on neuronal elements in the anterior pelvic ganglion of the male pig with immunohistochemistry and quantitative real-time PCR (qPCR). The ganglia were examined 3 and 6months after surgery. In 3-month-old castrated pigs (3MCP) 74% of adrenergic and 31% of cholinergic neurons stained for caspase-3 (CASP-3), and much greater numbers of perikarya than in the control animals expressed CGRP, galanin (GAL) and VIP (peptides known to have neuroprotective properties). In 6-months-old castrated pigs (6MCP), an excessive loss (90%) of neurons and intraganglionic nerve fibres was found. The survived adrenergic and cholinergic neurons also expressed CASP-3, CGRP, GAL or VIP. The qPCR results corresponded with immunofluorescence findings. In 3MCP, genes for CASP-3 and CGRP were up-regulated, while the expression of those for DβH, VAChT, GAL, VIP and SP displayed statistically insignificant variations. In 6MCP, distinctly up-regulated were genes for CGRP, GAL, VIP, SP, DβH and VAChT, while the expression of casp3 gene was down-regulated. The study revealed for the first time the excessive loss of pelvic neurons following castration, and a realistic assumption is proposed, that the neurons died due to apoptosis triggered by androgen deprivation.

309 Radiation-induced Schwann Cell Dedifferentiation Leads to Opposing Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons

Prostatic radiation therapy (RT) is a highly effective treatment for localized low-grade prostate cancer. Unfortunately, in 67-85% of patients, RT causes erectile dysfunction (ED). The direct molecular mechanisms underlying RT-induced ED are not understood, and no effective therapies have been identified. The major pelvic ganglia (MPG) in rats is anatomically equivalent to the hypogastric plexus in humans and can be used to assess radiation-induced nerve injury. Both whole and dissociated MPG cultures have examined neurite growth and apoptosis in models of ED; however, the impact of radiation on MPG neuron growth and survival has not been directly compared in these culture conditions. Determine the effects of ex vivo radiation on MPG neuron survival, growth, and gene expression in whole vs dissociated culture. MPGs from male Sprague-Dawley rats (n=14) were removed and irradiated ex vivo (0 or 800 cGy). For dissociated culture, MPGs were digested in collagenase/dispase. Neurons were collected by trituration and cultured on coverslips for 72 hours. Immunofluorescent staining for beta-tubulin III (Tubb3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and TUNEL assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole culture, MPGs were grown in Matrigel and neurite growth was measured at 72 hours. Gene expression of apoptotic markers (Caspase 1, 3), myelination (Sox2, Sox10, Krox20), glial dedifferentiation (GFAP), Tubb3, nNOS, and TH was measured in irradiated cultures of dissociated MPG neurons, isolated Schwann cells (SCs), and whole cultured MPGs by qPCR.

Embryonic stem cell-derived extracellular vesicle-mimetic nanovesicles rescue erectile function by enhancing penile neurovascular regeneration in the streptozotocin-induced diabetic mouse

Extracellular vesicles (EVs) have attracted particular interest in various fields of biology and medicine. However, one of the major hurdles in the clinical application of EV-based therapy is their low production yield. We recently developed cell-derived EV-mimetic nanovesicles (NVs) by extruding cells serially through filters with diminishing pore sizes (10, 5, and 1 μm). Here, we demonstrate in diabetic mice that embryonic stem cell (ESC)-derived EV-mimetic NVs (ESC-NVs) completely restore erectile function (~96% of control values) through enhanced penile angiogenesis and neural regeneration in vivo, whereas ESC partially restores erectile function (~77% of control values). ESC-NVs promoted tube formation in primary cultured mouse cavernous endothelial cells and pericytes under high-glucose condition in vitro; and accelerated microvascular and neurite sprouting from aortic ring and major pelvic ganglion under high-glucose condition ex vivo, respectively. ESC-NVs enhanced the expression of angiogenic and neurotrophic factors (hepatocyte growth factor, angiopoietin-1, nerve growth factor, and neurotrophin-3), and activated cell survival and proliferative factors (Akt and ERK). Therefore, it will be a better strategy to use ESC-NVs than ESCs in patients with erectile dysfunction refractory to pharmacotherapy, although it remains to be solved for future clinical application of ESC.

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.

MP59-08 OPPOSING EFFECTS OF NEURITE GROWTH IN WHOLE MAJOR PELVIC GANGLION CULTURE VS DISSOCIATED NEURON CULTURE FOLLOWING EX VIVO RADIATION

INTRODUCTION AND OBJECTIVES:Prostatic radiation therapy (RT) causes nerve damage to the major pelvic ganglia (MPG) resulting in erectile dysfunction (ED). To understand the impact of neuronal injur...

Negative Cumulative Impact of Low Testosterone and Cavernous Nerve Injury on Pelvic Neuron Survival is Improved by Testosterone Supplementation

Prostate cancer is often treated surgically with radical prostatectomy (RP). Following RP, erectile dysfunction (ED) occurs in 25–100% of men, negatively impacting their quality of life. RP induced ED is primarily attributed to injury of the cavernous nerves which branch from the major pelvic ganglia (MPG). Frequently men are 65 and older when diagnosed with prostate cancer and have low circulating testosterone (T) levels. Testosterone (T) is essential to maintaining neuronal health and to recovering nerve function. The objective of this study is to examine effects of RP‐induced cavernous nerve injury in an animal model of low T (castration) on MPG neuron survival and to assess if T supplementation can restore erectile function and MPG health.Male Sprague‐Dawley rats (12 weeks) were separated into control (no surgery; CON), castration (CAST), bilateral cavernous nerve injury (BCNI), CAST+BCNI (C+B), and CAST+BCNI and T supplementation (3mg/kg; C+B+T) groups (n=4/grp). After 4 weeks of CAST, rats underwent BCNI to mimic RP and the C+B+T group started T treatment for 2 weeks. At 18 weeks of age, erections were measured via stimulation of the cavernous nerve and direct measurement of intracavernosal to mean arterial pressure (ICP/MAP). Bilateral MPGs were collected, dissociated into neurons and cultured for 72 hours. Neurons were fixed and stained immunofluorescently for neuron‐specific class III beta‐tubulin to measure axonal branching and length and then co‐stained with TUNEL assay to identify apoptotic neurons. Additional neurons were stained with sympathetic (tyrosine hydroxylase) or nitrergic (neuronal nitric oxide synthase) markers.Overall, CAST, BCNI and C+B decreased ICP/MAP, neurite growth and branching, increased apoptosis, elevated sympathetic neuron populations, and reduced nitrergic neurons compared to CON (p&lt;0.05). CAST and BCNI alone both caused 25% decrease in neurite length, 20% decrease in branching and 2‐fold increase in apoptosis (p&lt;0.05 vs CON). However, C+B caused a greater decrease in neurite length by 33%, decreased branching by 50% and doubled apoptosis (p&lt;0.05 vs CON). The population of pro‐erectile nitrergic neurons were reduced by 60% in the CAST or BCNI alone and decreased by 70% in the C+B (p&lt;0.05 vs CON). In contrast, the anti‐erectile sympathetic neurons were increased 30% with BCNI and 40% C+B (p&lt;0.05 vs CON). Supplementation with T restored neurite length in C+B but did not improve neurite branching. Neuronal apoptosis was reduced 50% in C+B+T; however was still elevated above CON (p&lt;0.05). T supplementation restored nitrergic and sympathetic neuron populations to control levels. In addition, T restored erectile function as measured by ICP/MAP in C+B rats (p&lt;0.05).Nerve injury in a low androgen state (C+B) markedly impaired neuritogenesis and caused a greater decrease in nitrergic, pro‐erectile neurons compared to CON, CAST or BCNI alone. These data indicate that recovery of erectile function following RP‐induced nerve damage in a state of low T is unlikely and will lead to a higher incidence and severity of ED. T improves both erectile function and neuronal health, and should be considered for prostate cancer survivors.Support or Funding InformationSMSNA, SNCURCS, ECU URCAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse

IntroductionPenile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in diabetic patients, which causes poor response to oral phosphodiesterase-5 inhibitors. Nerve growth factor precursor (proNGF) and its p75 neurotrophin receptor (p75NTR) have been known to be involved in microvascular complications and neurodegeneration. AimTo examine the role of proNGF and its receptor p75NTR signaling pathway in diabetic ED, and to determine the effectiveness of proNGF neutralizing antibody (proNGF-Ab) in restoring erectile function in streptozotocin (STZ)-induced diabetic mice. MethodsDiabetes mellitus was induced by intraperitoneal injection of STZ (50 mg/kg) into 8-week-old C57BL/6 male mice for 5 consecutive days. At 8 weeks after the induction of diabetes mellitus, the animals were distributed into 3 groups: controls and STZ-induced diabetic mice receiving 2 intracavernous injections of either saline (days −3 and 0; 20 μL) or proNGF-Ab (days −3 and 0; 20 μg in 20 μL of saline). We also examined the effect of proNGF-Ab or p75NTR small interfering RNA in primary cultured mouse cavernous endothelial cells, pericytes, and major pelvic ganglion. Main Outcome MeasuresErectile function was measured by electrical stimulation of the cavernous nerve at 2 weeks after treatment, and the penis was then harvested for histologic and biochemical studies. ResultsThe cavernous expression of proNGF and p75NTR was upregulated under diabetic conditions. Intracavernous injection of proNGF-Ab successfully restored erectile function in diabetic mice, which reach 93–96% of control values. ProNGF-Ab significantly restored cavernous endothelial cell, pericyte, and neuronal cell content, and increased endothelial cell-to-cell junction proteins in the diabetic mice. Under the high-glucose condition, proNGF-Ab or p75NTR small interfering RNA promoted tube formation in mouse cavernous endothelial cells and pericytes, decreased apoptosis of endothelial cells and pericytes, and enhanced neurite sprouting from major pelvic ganglion. Clinical ImplicationsThe ProNGF/p75NTR pathway will be a new therapeutic target for diabetic ED. Strength & LimitationsThis is the first study demonstrating the efficacy of the inhibition of proNGF/p75NTR pathway in diabetic ED. Further studies are needed to test whether a different dosing of proNGF-Ab would induce more durable erectile function recovery. ConclusionOur findings suggest that inhibition of the proNGF/p75NTR signaling pathway is a promising therapeutic strategy for diabetic ED.Nguyen NM, Song K-M, Choi M-J, et al. Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse. J Sex Med 2019;16:351–364.

Insulin-Like Growth Factor-1–Loaded Polymeric Poly(Lactic-Co-Glycolic) Acid Microspheres Improved Erectile Function in a Rat Model of Bilateral Cavernous Nerve Injury

BackgroundPrevious studies have documented improvement in erectile function after bilateral cavernous nerve injury (BCNI) in rats with the use of pioglitazone. Our group determined this improvement to be mediated by the insulin-like growth factor-1 (IGF-1) pathway. AimTo eliminate the systemic effects of pioglitazone and evaluate the local delivery of IGF-1 by polymeric microspheres after BCNI in the rat. MethodsMale Sprague–Dawley rats aged 10–12 weeks were assigned at random to 3 groups: sham operation with phosphate buffered saline (PBS)-loaded microspheres (sham group), crush injury with PBS-loaded microspheres (crush group), and crush injury with IGF-1–loaded microspheres (IGF-1 group). Poly(lactic-co-glycolic) acid microspheres were injected underneath the major pelvic ganglion (MPG). IGF-1 was released at approximately 30 ng/mL/day per MPG per rat. OutcomesFunctional results were demonstrated by maximal intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP). Protein-level analysis data of IGF-1 receptor (IGF-1R), extracellular signal–regulated kinase (ERK)-1/2, and neuronal nitric oxide synthase (nNOS) were obtained using Western blot analysis and immunohistochemistry for both the cavernosal tissue and the MPG and cavernous nerve (CN). ResultsAt 2 weeks after nerve injury, animals treated with IGF-1 demonstrated improved erectile functional recovery (ICP/MAP) at all voltages compared with BCNI (2.5V, P = .001; 5V, P < .001; 7.5V, P < .001). Western blot results revealed that up-regulation of the IGF-1R and ERK-1/2 in both the nervous and erectile tissue was associated with improved erectile function recovery. There were no significant between-group differences in nNOS protein levels in cavernosal tissue, but there was an up-regulation of nNOS in the MPG and CN. Immunohistochemistry confirmed these trends. Clinical TranslationLocal up-regulation of the IGF-1R in the neurovascular bed at the time of nerve injury may help men preserve erectile function after pelvic surgery, such as radical prostatectomy, eliminating the need for systemic therapy. Strengths & LimitationsThis study demonstrates that local drug delivery to the MPG and CN can affect the CN tissue downstream, but did not investigate the potential effects of up-regulation of the growth factor receptors on prostate cancer tissue. ConclusionStimulating the IGF-1R at the level of the CN has the potential to mitigate erectile dysfunction in men after radical prostatectomy, but further research is needed to evaluate the safety of this growth factor in the setting of prostate cancer.Haney NM, Talwar S, Akula PK, et al. Insulin-Like Growth Factor-1–Loaded Polymeric Poly(Lactic-Co-Glycolic) Acid Microspheres Improved Erectile Function in a Rat Model of Bilateral Cavernous Nerve Injury. J Sex Med 2019;16:383–393.

The expression of androgen receptor in neurons of the anterior pelvic ganglion and celiac-superior mesenteric ganglion in the male pig.

The present study investigated the expression of androgen receptor (AR) in neurons of the anterior pelvic ganglion (APG) and celiac-superior mesenteric ganglion (CSMG; ganglion not involved in the innervation of reproductive organs) in the male pig with quantitative real-time PCR (qPCR) and immunohistochemistry. qPCR investigations revealed that the level of AR gene expression in the APG tissue was approximately 2.5 times higher in the adult (180-day-old) than in the juvenile (7-day-old) boars. Furthermore, in both the adult and juvenile animals it was sig- nificantly higher in the APG than in CSMG tissue (42 and 85 times higher, respectively). Immu- nofluorescence results fully confirmed those obtained with qPCR. In the adult boars, nearly all adrenergic (DβH-positive) and the majority of non-adrenergic neurons in APG stained for AR. In the juvenile animals, about half of the adrenergic and non-adrenergic neurons were AR-posi- tive. In both the adult and juvenile animals, only solitary CSMG neurons stained for AR. The present results suggest that in the male pig, pelvic neurons should be considered as an element of highly testosterone-dependent autonomic circuits involved in the regulation of urogenital func- tion, and that their sensitization to androgens is a dynamic process, increasing during the prepu- bertal period.

Urethral regions with differential tissular composition may underlie urinary continence and voiding function in female rats.

To analyze, in female rats, the anatomical and histological features of the urethra and its relationship with the vagina and clitoris, and its innervation. Seventeen adult female Wistar rats were used. Gross anatomy and acetylcholinesterase (AchE) histochemistry were performed to describe the urethral features, adjacent structures, and innervation. The histomorphometric characteristics of the urethra were determined in transversal, longitudinal, or coronal sections stained with Masson's Trichrome. The female rat urethra is not a homogeneous tubular organ. The pre-pelvic and pelvic regions are firmly attached to the vagina with belt-like striated fibers forming a urethra-vaginal complex. The bulbar regions have curved segments and a narrow lumen. The clitoral region is characterized by a urethra-clitoral complex surrounded by a vascular plexus. The lumen area and thickness of the urethral layers significantly varied between regions (P < 0.05). Innervation of the urethra arrives from the major pelvic ganglion, the dorsal nerve of the clitoris (DNC), and the motor branch of the sacral plexus (MBSP). Differential tissular composition of the urethra may underlie urinary continence and voiding dysfunction through different physiological mechanisms. The urethra-vagina complex seems to be the main site controlling urinary continence through active muscular mechanisms, while the bulbar urethra provides passive mechanisms and the urethra-clitoris complex seems to be crucial for distal urethral closure by means of a periurethral vascular network.

Functional and molecular characterisation of the bilateral pelvic nerve crush injury rat model for neurogenic detrusor underactivity.

To create a rat model for neurogenic detrusor underactivity (DU) by bilateral pelvic nerve crush injury (BPNI) and to study temporal changes in detrusor contractility and morphology. Male Sprague-Dawley rats were subjected to BPNI or sham surgery and evaluated at 1, 3 and 9weeks after surgery. Bladder function was determined invivo by awake cystometry, micturition pattern analysis, and 24-h urine collection. Bladders were harvested for invitro pharmacological investigation by isometric tension recording. Bladders and major pelvic ganglia were investigated by quantitative reverse transcription-polymerasechain reaction and histochemistry. Overflow incontinence was observed at 1week after BPNI. At 3 and 9weeks after BPNI, rats showed a bladder phenotype characteristic for DU with increased post-void residual urine volumes, reduced voiding efficiencies, and lower maximum pressures. In isolated bladder strips, contractile responses to KCl, carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-mATP) were preserved. On the other hand, neural-induced contractility was reduced after BPNI, in line with reduced expression of protein gene product 9.5 and choline acetyltransferase in the major pelvic ganglion at 1week after BPNI. The bladder-to-body weight ratio and detrusor thickness increased after BPNI, indicating detrusor hypertrophy to compensate for the reduced neural input. BPNI induces a rat model for neurogenic DU. In this model, the detrusor maintains its contractility but denervation of the detrusor was observed.

Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth

BackgroundErectile dysfunction (ED) is common following radiation therapy (RT) for prostate cancer. Although the cause of RT-induced ED is unknown, damage to both the neuronal and vascular components supporting erections are often implicated. AimTo determine the effects of prostatic RT on erections, penile vascular physiology, and major pelvic ganglia (MPG) neuron growth and survival in a rat model. MethodsMale rats underwent 0 Gy or 22 Gy single fraction of prostate-confined, conformal RT. At 2 weeks or 10 weeks post-RT (n = 10/group), cavernous nerve stimulation was performed and erections were assessed. Tissue bath experiments were performed to assess both penile artery and internal pudendal artery (IPA) function. MPGs were dissociated and neurons grown in culture for 72 hours. Immunofluorescence staining was done to quantify neuron survival (terminal deoxynucleotidyl transferase nick-end labeling), outgrowth (beta-tubulin III), type (nitric oxide synthase [nNOS] and tyrosine hydroxylase [TH]), and nerve injury markers (small GTPase Rac1 and ninjurin-1 [Ninj-1]). Whole MPG real-time quantitative polymerase chain reaction (qPCR) was performed to measure expression of genes related to nerve type, neuron injury, repair, and myelination, such as Ninj-1, Rac1, ATF3, GAP43, GFAP, SOX10, and KROX20. OutcomesIntracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio, smooth muscle contractility and relaxation, gene expression, neuritogenesis, and apoptosis. ResultsFollowing RT, ICP/MAP was unchanged at 2 weeks or 10 weeks. Nerve-mediated penile contraction was increased at 2 weeks, whereas adrenergic contraction was reduced at 10 weeks. Penile relaxation and IPA vasoreactivity were unchanged. Neuronal apoptosis was more than doubled both early and late post-RT. RT caused a progressive decrease in neurite branching but an early increase and then late decrease in neurite lengthening. RT reduced the numbers of nNOS-positive neurons both early and late and also decreased MPG nitrergic gene expression. TH neurons and gene expression were unchanged at 2 weeks; however, both were decreased after 10 weeks. Although most markers of gene injury and repair were unaffected early post-RT, MPG expression of Ninj1 and GFAP increased. After 10 weeks, Ninj1 and GFAP remained elevated while markers of neuron injury (ATF3), outgrowth (GAP43 and Rac1), and myelin regulation (SOX10) were decreased. Clinical TranslationRT-induced ED may result from damage to the ganglia controlling erections. Strengths & LimitationsThis study used a clinically relevant, prostate-confined model to examine neurovascular structures not accessible in human studies. Unfortunately, rats did not exhibit ED at this time point. ConclusionThis is the first study to demonstrate impaired health and regeneration potential of dissociated MPG neurons following RT. Neuronal injury was apparent early post-RT and persisted or increased over time but was insufficient to cause ED at the time points examined.Powers SA, Odom MR, Pak ES, et al. Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth. J Sex Med 2019;16:27–41.

Transplantation of induced pluripotent stem cell-derived mesenchymal stem cells improved erectile dysfunction induced by cavernous nerve injury.

Erectile dysfunction (ED) is an important kind of postoperative complication of pelvic surgery that affects patients' quality of life. Transplantation of mesenchymal stem cells (MSC) has been found to alleviate ED caused by cavernous nerve injury (CNI) in rats. However, little is known about whether induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) have a therapeutic effect on CNI ED. We established an ED model on rats and evaluated the effect of iMSC on it.Methods: Eight-week-old male Sprague-Dawley rats were assigned to four groups and received following operation: sham operation (sham group); bilateral CNI and phosphate-buffered saline (PBS) injections (PBS group); bilateral CNI and adipose-derived mesenchymal stem cells transplantation (adMSC group); or bilateral CNI and iMSC injection (iMSC group). After therapy, the cavernous nerve was stimulated by electricity and the intracavernous pressure (IAP)/mean arterial blood pressure (MAP) was measured. The endothelial and smooth muscle tissue in the penis was assessed histologically with Masson's trichrome stain. Immunofluorescence/immunohistochemical stains were applied for the detection of nNOS, vWF, eNOS, SMA, Desmin, S100β, and caspase-3. Nude rats CNI ED model was established for the evaluation of iMSC longevity and differentiation capacity. The paracrine factors were assessed by real-time PCR.Results: Transplantation of iMSC significantly restored the IAP/MAP in this CNI ED model and showed long-term effects. It could rescue the expression of vWF, eNOS, SMA, and Desmin, which indicated the alleviation of endothelial and smooth muscle tissues of the penis. iMSC therapy also could increase the expression of nNOS in the cavernosum and S100β in the major pelvic ganglia (MPG) which contributed to the erectile function. Moreover, the level of BAX and caspase-3 were reduced and Bcl-2 was increased, which indicated the anti-apoptosis effects of iMSC. The iMSC showed little transdifferentiation and exerted their function by activating the secretome of the host.Conclusion: Transplantation of iMSC significantly improved ED induced by CNI. The iMSC may exert their effects via paracrine factors and may be a promising therapeutic candidate for treating CNI ED in the future.

Neural interrelationships of autonomic ganglia from the pelvic region of male rats.

The aims of the present study were to describe, in male rats, the anatomical organization of the major and accessory pelvic ganglia (MPG, AG; respectively), the interrelationship of the pelvic plexus components, and the morphometry of the pelvic postganglionic neurons. Anatomical, histochemical and histological studies were performed in anesthetized adult Wistar male rats. We found that the pelvic plexus consists of intricate neural circuits composed of two MPG, and three pairs of AG (AGI, AGII, AGIII) anatomically interrelated through ipsilateral and contralateral commissural nerves. Around 30 nerves emerge from each MPG and 17 from AGI and AGII. The MPG efferent nerves spread out preganglionic information to several pelvic organs controlling urinary, bowel, reproductive and sexual functions, while AG innervation is more regional, and it is confined to reproductive organs located in the rostral region of the urogenital tract. Both MPG and AG contain nerve fascicles, blood vessels, small intensely fluorescent cells, satellite cells and oval neuronal somata with one to three nucleoli. The soma area of AG neurons is larger than those of MPG neurons (p < 0.005). The MPG contains about 75% of the total pelvic postganglionic neurons. Our findings corroborated previous reports about MPG inputs, and add new information regarding pelvic ganglia efferent branches, AG neurons (number and morphometry), and neural interrelationship between the pelvic plexus components. This information will be useful in designing future studies about the role of pelvic innervation in the physiology and pathophysiology of pelvic functions.

Pelvic and hypogastric nerves are injured in a rat prostatectomy model, contributing to development of stress urinary incontinence

Urinary incontinence affects 40% of elderly men, is common in diabetic patients and in men treated for prostate cancer, with a prevalence of up to 44%. Seventy-two percent of prostatectomy patients develop stress urinary incontinence (SUI) in the first week after surgery and individuals who do not recover within 6 months generally do no regain function without intervention. Incontinence has a profound impact on patient quality of life and a critical unmet need exists to develop novel and less invasive SUI treatments. During prostatectomy, the cavernous nerve (CN), which provides innervation to the penis, undergoes crush, tension, and resection injury, resulting in downstream penile remodeling and erectile dysfunction in up to 85% of patients. There are other nerves that form part of the major pelvic ganglion (MPG), including the hypogastric (HYG, sympathetic) and pelvic (PN, parasympathetic) nerves, which provide innervation to the bladder and urethra. We examine if HYG and PNs are injured during prostatectomy contributing to SUI, and if Sonic hedgehog (SHH) regulatory mechanisms are active in the PN and HYG nerves. CN, PN, HYG and ancillary (ANC) of uninjured, sham and CN crush/MPG tension injured (prostatectomy model) adult Sprague Dawley rats (n = 37) were examined for apoptosis, sonic hedgehog (SHH) pathway, and intrinsic and extrinsic apoptotic mechanisms. Fluorogold tracing from the urethra/bladder was performed. PN and HYG response to SHH protein was examined in organ culture. TUNEL, immunohistochemical analysis for caspase-3 cleaved, -8, -9, SHH, Patched and Smoothened (SHH receptors), and neurite formation, were examined. Florogold positive neurons in the MPG were reduced with CN crush. Apoptosis increased in glial cells of the PN and HYG after CN crush. Caspase 9 was abundant in glial cells (intrinsic), while caspase-8 was not observed. SHH and its receptors were abundant in neurons and glia of the PN and HYG. SHH treatment increased neurite formation. PN and HYG injury occur concomitant with CN injury during prostatectomy, likely contributing to SUI. PN and HYG response to SHH treatment indicates an avenue for intervention to promote regeneration and prevent SUI.

Sonic hedgehog regulation of cavernous nerve regeneration and neurite formation in aged pelvic plexus

IntroductionErectile dysfunction (ED) is a significant health concern that greatly impacts quality of life, and is common in men as they age, impacting 52% of men between the ages of 40 and 70. A significant underlying cause of ED development is injury to the cavernous nerve (CN), a peripheral nerve that innervates the penis. CN injury also occurs in up to 82% of prostatectomy patients. We recently showed that Sonic hedgehog (SHH) protein delivered by peptide amphiphile (PA) nanofiber hydrogel to the CN and penis of a prostatectomy model of CN injury, is neuroprotective, accelerates CN regeneration, improves erectile function ~60%, preserves penile smooth muscle 56% and suppresses collagen deposition 30%. This regenerative potential is substantial in an adult prostatectomy model (P120). However prostatectomy patients are typically older (61.5 ± 9.6 years) and our models should mimic patient conditions more effectively when considering translation. In this study we examine regenerative potential in an aged prostatectomy model (P200–329). MethodsThe caudal portion of the pelvic ganglia (MPG) and CN were dissected from adult (n = 11), and aged (n = 13) Sprague Dawley rats, and were grown in organ culture 3 days. Uninjured and 2 day CN crushed MPG/CN were exposed to Affi-Gel beads containing SHH protein, PBS (control), or 5e1 SHH inhibitor. Neurites were quantified by counting the number of growth cones normalized by tissue perimeter (mm) and immunohistochemistry for SHH, patched1 (PTCH1), smoothened (SMO), GLI1-3, and GAP43 were performed. ResultsSHH treatment increased neurites 3.5-fold, in uninjured adult, and 5.7-fold in aged rats. Two days after CN crush, SHH treatment increased neurites 1.8-fold in adult rats and 2.5-fold in aged rats. SHH inhibition inhibited neurite formation in uninjured MPG/CN but not in 2 day CN crushed MPG/CN. PTCH1 and SMO (SHH receptors), and SHH transcriptional activators/repressors, GLI1-3, were abundant in aged MPG/CN with unaltered localization. ROCK1 was induced with SHH treatment. ConclusionsReintroduction of SHH protein in an aged prostatectomy model is even more effective in promoting neurite formation/CN regeneration than in the adult. The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. These studies are significant because most prostatectomy patients are not young and healthy, as with adult rats, so the aged prostatectomy model will more accurately simulate ED patient response. Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients.