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

INTRODUCTION: The 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. AIM: To 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. METHODS: Human 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 MEASURE: The main outcome measure includes intracavernous pressure/mean arterial pressure ratio, neural, endothelial, smooth muscle cell regeneration, mKATE fluorescence, and IVIS imaging. RESULTS: The 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 IMPLICATIONS: Placenta-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 & LIMITATIONS: Single 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. CONCLUSION: Human 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.

Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth.

BACKGROUND: Erectile 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. AIM: To determine the effects of prostatic RT on erections, penile vascular physiology, and major pelvic ganglia (MPG) neuron growth and survival in a rat model. METHODS: Male 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. OUTCOMES: Intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio, smooth muscle contractility and relaxation, gene expression, neuritogenesis, and apoptosis. RESULTS: Following 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 TRANSLATION: RT-induced ED may result from damage to the ganglia controlling erections. STRENGTHS & LIMITATIONS: This 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. CONCLUSION: This 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.

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.

BACKGROUND: Previous 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. AIM: To eliminate the systemic effects of pioglitazone and evaluate the local delivery of IGF-1 by polymeric microspheres after BCNI in the rat. METHODS: Male 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. OUTCOMES: Functional 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). RESULTS: At 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 TRANSLATION: Local 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 & LIMITATIONS: This 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. CONCLUSION: Stimulating 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.

M1 Macrophages Are Predominantly Recruited to the Major Pelvic Ganglion of the Rat Following Cavernous Nerve Injury.

INTRODUCTION: Neurogenic erectile dysfunction is a common sequela of radical prostatectomy. The etiology involves injury to the autonomic cavernous nerves, which arise from the major pelvic ganglion (MPG), and subsequent neuroinflammation, which leads to recruitment of macrophages to the injury site. Currently, two macrophage phenotypes are known: neurotoxic M1 macrophages and neuroprotective M2 macrophages. AIM: To examine whether bilateral cavernous nerve injury (BCNI) in a rat model of erectile dysfunction would increase recruitment of neurotoxic M1 macrophages to the MPG. METHODS: Male Sprague-Dawley rats underwent BCNI and the MPG was harvested at various time points after injury. The corpora cavernosa was used to evaluate tissue myographic responses to electrical field stimulation ex vivo. Quantitative real-time polymerase chain reaction was used to examine the gene expression of global macrophage markers, M1 macrophage markers, M2 macrophage markers, and cytokines and chemokines in the MPG. Mathematical calculation of the M1/M2 index was used to quantify macrophage changes temporally. Western blot of MPG tissues was used to evaluate the protein amount of M1 and M2 macrophage markers quantitatively. Immunohistochemistry staining of MPGs for CD68, CD86, and CD206 was used to characterize M1 and M2 macrophage infiltration. MAIN OUTCOME MEASURES: Corpora cavernosa responsiveness ex vivo; gene (quantitative real-time polymerase chain reaction) and protein (western blot) expressions of M1 and M2 markers, cytokines, and chemokines; and immunohistochemical localization of M1 and M2 macrophages. RESULTS: BCNI impaired the corporal parasympathetic-mediated relaxation response to electrical field stimulation and enhanced the contraction response to electrical field stimulation. Gene expression of proinflammatory (Il1b, Il16, Tnfa, Tgfb, Ccl2, Ccr2) and anti-inflammatory (Il10) cytokines was upregulated in the MPG 48 hours after injury. M1 markers (CD86, inducible nitric oxide synthase, interleukin-1ß) and M2 markers (CD206, arginase-1, interleukin-10) were increased after BCNI in the MPG, with the M1/M2 index above 1.0 indicating that more M1 than M2 macrophages were recruited to the MPG. Protein expression of the M1 macrophage marker (inducible nitric oxide synthase) was increased in MPGs after BCNI. However, the protein amount of M2 macrophage markers (arginase-1) remained unchanged. Immunohistochemical characterization demonstrated predominant increases in M1 (CD68+CD86+) macrophages in the MPG after BCNI. CONCLUSION: These results suggest that an increase in M1 macrophage infiltration of the MPG after BCNI is associated with impaired neurogenically mediated erectile tissue physiology ex vivo and thus has significant implications for cavernous nerve axonal repair. Future studies are needed to demonstrate that inhibition of M1 macrophage recruitment prevents erectile dysfunction after CNI.

GGF2 is neuroprotective in a rat model of cavernous nerve injury-induced erectile dysfunction.

INTRODUCTION: Erectile dysfunction is a major complication of radical prostatectomy, commonly associated with penile neuropathy. In animal models of peripheral nerve injury, glial growth factor-2 (GGF2), a member of the neuregulin family of growth factors, has neuroprotective and neurorestorative properties, but this potential has not been established after cavernous nerve (CN) injury. AIMS: The effectiveness of GGF2 in preserving axonal integrity and recovering erectile function in a rat model of radical prostatectomy-associated CN injury. METHODS: Adult male Sprague-Dawley rats underwent bilateral CN crush injury (BCNI) or sham surgery. Rats were administered GGF2 (0.5, 5, or 15 mg/kg) or vehicle subcutaneously 24 hour pre and 24-hour post-BCNI, and once weekly for 5 weeks. Erectile function was assessed in response to electrical stimulation of the CN. CN survival was assessed by fluorogold retrograde axonal tracing in major pelvic ganglia (MPG). Unmyelinated axons in the CNs were quantitated by electron microscopy. MAIN OUTCOME MEASURES: Erectile function recovery, CN survival, and unmyelinated CN axon preservation in response to GGF2 treatment following BCNI. RESULTS: Erectile function was decreased (P < 0.05) after BCNI, and it was improved (P < 0.05) by all doses of GGF2. The number of fluorogold-labeled cells in the MPG was reduced (P < 0.05) by BCNI and was increased (P < 0.05) by GGF2 (0.5 and 5 mg/kg). The percentage of denervated Schwann cells in the BCNI group was higher (P < 0.05) than that in the sham-treated group and was decreased (P < 0.05) in the GGF2-treated (5 mg/kg) BCNI group. In the BCNI + GGF2 (5 mg/kg) group, the unmyelinated fiber histogram demonstrated a rightward shift, indicating an increased number of unmyelinated axons per Schwann cell compared with the BCNI group. CONCLUSIONS: GGF2 promotes erectile function recovery following CN injury in conjunction with preserving unmyelinated CN fibers. Our findings suggest the clinical opportunity to develop GGF2 as a neuroprotective therapy for radical prostatectomy.

Intravenous Preload of Mesenchymal Stem Cells Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury.

INTRODUCTION: We evaluated the potential preventive effects and mechanisms of intravenously preloaded mesenchymal stem cells (MSCs) for erectile dysfunction (ED) in a cavernous nerve (CN) injury model. METHODS: Male Sprague-Dawley (SD) rats were used for this study. Rats were randomized into two groups. One group was intravenously preloaded with MSCs (1.0 × 10(6) cells in 1 mL total fluid volume) and the other was infused with medium alone (1 mL Dulbecco's modified Eagle's medium [DMEM]) for sham control, respectively. Crushed CN injury was induced immediately after infusion. The surgeon was blind to the experimental conditions (MSC or medium). MAIN OUTCOME MEASURES: To assess erectile function, we measured the intracavernous pressure (ICP) and arterial pressure (AP) at 1 hour and 2 weeks after CN injury. After measuring the initial ICP/AP of pre-injury (normal) male SD rats, they were randomized into the two groups and infused with MSCs or medium. PKH26-labelled MSCs were used for tracking. To investigate the mRNA expression levels of neurotrophins in the major pelvic ganglia (MPG), we performed real-time quantitative real-time polymerase chain reaction. RESULTS: The reduction of ICP/AP and area under the curve of ICP (ICP-AUC) in the MSC group was significantly lower than in the DMEM group (P < 0.05; P < 0.05) at 1 hour. The ICP/AP and ICP-AUC at 2 weeks post-injury in the MSC group was significantly higher than in the DMEM group (P < 0.01; P < 0.05). The preloaded PKH26-labelled MSCs were detected in the MPG and CN using confocal microscopy indicating homing of the cells to the injured nerve and ganglia. Glia cell-derived neurotrophic factor (GDNF) and neurturin, which are important neurotrophic factors for erection, had expression levels in MPG significantly higher in the MSC group than in the DMEM group (P < 0.01, 0.05). CONCLUSION: Intravenous preload of MSCs before a CN injury may prevent or reduce experimental ED.

Decrease in neuronal nicotinic acetylcholine receptor subunit and PSD-93 transcript levels in the male mouse MPG after cavernous nerve injury or explant culture.

Quantitative real-time PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic ACh receptor (nAChR) subunit and postsynaptic density (PSD)-93 transcript levels. Subunits α3, ß4, and α7, commonly expressed in the MPG, were selected for analysis. After 72 h in explant culture, MPG transcript levels for α3, ß4, α7, and PSD-93 were significantly depressed. Three days after cavernous nerve axotomy or crush in vivo, transcript levels for α3, ß4, and PSD-93, but not for α7, were significantly depressed. Three days after dissection of the cavernous nerve free of underlying tissue and application of a 5-mm lateral stretch (manipulation), transcript levels for α3 and PSD-93 were also significantly decreased. Seven days after all three surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only after axotomy or nerve crush. At 30 days postsurgery, transcript levels for the nAChR subunits and PSD-93 had recovered. ACh-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence showing that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a downregulation of nAChR subunit and PSD-93 expression after cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs after pelvic surgeries.

Whole genome microarray of the major pelvic ganglion after cavernous nerve injury: new insights into molecular profile changes after nerve injury.

UNLABELLED: What's known on the subject? and What does the study add? With the present study, we aimed to provide a global picture of the molecular processes that are activated by CN injury. The present study used genomic expression profiling to identify candidate genes that might be useful targets in the CN recovery process and, thus, the ultimate preservation of penile erection. Regeneration of the CN and axonal outgrowth clearly involve changes in multiple biochemical pathways that have never been investigated by microarray analysis. We analyzed global gene expression in the major pelvic ganglion at early stages (48 h and 14 days) after CN injury and focused on the detection of changes in genes related to nervous tissue repair and proliferation. The findings of the present study provide important insight into the molecular systems affected by CN injury and identify candidate genes that may be utilized for novel molecular-based therapies for the preservation and protection of the CN during RP. OBJECTIVES: To to examine the complexity of the many molecular systems involved in supporting cavernous nerve (CN) repair and regeneration in a rat model of bilateral crush injury utilizing a microarray analysis approach. Erectile dysfunction (ED) is a common clinical complication after prostate cancer treatment by radical prostatectomy, and recovery of erectile function can take as long as 2 years. There are gaps in our understanding of the autonomic pelvic innervation of the penis that still need to be addressed for the development of an adequate treatment strategy for post-prostatectomy ED. The molecular mechanisms of the intrinsic ability of CN to regenerate after an injury have not been elucidated. MATERIALS AND METHODS: We analyzed global gene expression in the major pelvic ganglion 48 h and 14 days after CN injury. Overall, a comparative analysis showed that 325 genes changed at the 48-h time point and 114 genes changed at 14 days. There were 60 changed genes in common with both time points. Using the Ingenuity Pathway Analysis® system (Ingenuity Systems, Inc., Redwood City, CA, USA), we were able to analyze the significantly changed genes that were unique and common to each time point by biological function. We focused on the detection of changes related to nervous tissue repair and proliferation, molecular networks of neurotrophic factors, stem cell regulation and synaptic transmission. RESULTS: There was strong evidence of the early mobilization of genes involved in repair and neuroprotection mechanisms (SERPINF1, IGF1, PLAU/PLAUR, ARG1). Genes related to nervous system development (ATF3 GJA1, PLAU, SERPINE1), nerve regeneration (SERPINE2, IGF1, ATF3, ARG1) and synaptic transmission (GJC1, GAL) were changed. Several genes related to proliferation as well as apoptosis (A2M, ATF3, C3, EGR4, FN1, GJA1, GAL) were also changed, possibly as part of a protective mechanism or the initiation of remodelling. CONCLUSIONS: The results obtained show that multiple biological processes are associated with injury and repair of the CN and provide a systematic genome-wide screen for neurotrophic and/or inhibitory pathways of nerve regeneration. These data identify the candidate genes that may be utilized in novel molecular-based therapies for the preservation and protection of the CN during radical prostatectomy.

Proportional release of norepinephrine and dopamine- -hydroxylase from sympathetic nerves.

Dopamine-beta-hydroxylase(DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, is localized in the vesicles containing catecholamine in sympathetic nerves. This enzyme is released with norepinephrine when the nerves to the guinea pig vas deferens are stimulated in vitro, and the amount of enzyme discharged increases as the length of stimulation periods increases. The amount of DBH released is proportional to the amount of norepinephrine released, and the ratio of norepinephrine to DBH discharged into the incubation medium is similar to that in the soluble portion of the contents of the synaptic vesicles from the vas deferens. These data are compatible with the release of the neurotransmitter norepinephrine and DBH from symnpathetic nerves by a process of exocytosis.

Magnifying lenses assisted nerve-sparing radical hysterectomy and prevention of nerve plexus trauma.

The objectives of the study were to present a new approach for nerve-sparing radical hysterectomy (NSRH) with the assistance of magnifying lenses and to describe the differences in autonomic nerve plexus trauma between NSRH type III and conventional radical hysterectomy (RH) types II and III with the aid of immunohistochemistry. Eighteen women with FIGO stage IB(1)-IB(2) cervical cancer underwent loupes-assisted NSRH (n = 8), RH type II (n = 6), and RH type III (n = 4). Biopsies were taken intraoperatively from uterosacral ligament (USL) and cardinal ligament (CL), as well as from anterior vaginal wall (AVW) and posterior vaginal wall (PVW). Immunohistochemistry was approached with the use of S-100 protein, a general nerve marker. The percentage area of immunoreactivity (PAI) was used as an objective quantitative measure of nerve fibers within the ligaments. The PAI was greater in RH-III biopsies from both USL and CL (P < 0.001) when compared with RH-II and NSRH biopsies. For AVW and PVW, PAI differences were not statistically significant (AVW, P = 0.119; PVW, P = 0.067). Uterine-supporting ligaments represent a major pathway for autonomic nerves to the pelvic organs. As significantly more autonomic nerves are transected during the division of the uterine-supporting ligaments in RH type III, a more careful approach in the dissection of the ligaments through nerve-preserving techniques seems to be necessary in order to prevent iatrogenic intraoperative injury of the pelvic plexus and reduce or prevent postoperative complications.

Anatomical and physiological properties of pelvic ganglion neurons in female mice.

Most neurons that regulate motility and blood flow in female pelvic organs are located within pelvic (paracervical) ganglia. In this study we investigated the anatomical and physiological properties of neurons within mouse (C57/Bl/6) paracervical ganglia. Most neurons showed immunoreactivity for choline acetyl transferase (CHAT) and were presumably cholinergic. Few neurons (approximately 5%) were tyrosine hydroxylase (TH) positive. Immunohistochemical labelling for microtubule associated protein 2 showed most neurons had small somata (cross sectional area approximately 300 microm(2)) and lacked dendrites. Action potential (AP) discharge characteristics, determined by depolarising current step injection, revealed most neurons (70%) adapted rapidly to depolarising current injection and were classified as "phasic". The remaining neurons discharged APs throughout the current step and were classified as "tonic". Membrane properties and current-voltage relationships were similar in phasic and tonic neurons, however the afterhyperpolarisation was significantly smaller in tonic neurons. Stimulation of preganglionic axons usually evoked a single strong preganglionic input (21/27 and 9/10 for pelvic and hypogastric nerves, respectively). In 19 preparations where we tested for inputs from both nerves pelvic inputs predominated (23/45 neurons) and inputs via the hypogastric nerve were rarely observed (3/45 neurons). Together, our data indicate that most neurons within mouse paracervical ganglia are cholinergic and parasympathetic. As there is little anatomical or functional evidence for integration of preganglionic inputs we propose that the role of paracervical neurons is restricted to one of spatial amplification or filtering of preganglionic inputs.

Androgen and estrogen receptor-mediated mechanisms of testosterone action in male rat pelvic autonomic ganglia.

Although male reproductive function is primarily androgen dependent, many studies suggest that estrogens have direct actions on the male reproductive organs. Pelvic autonomic neurons provide the motor control of the internal reproductive organs and the penis and various properties of these neurons are affected by endogenous androgens. However, the possible role of estrogens at this site has not been examined. Here we have investigated the significance of estrogens produced by aromatization of testosterone (T) in the physiological actions of androgens on adult male rat pelvic ganglion neurons. Reverse transcriptase polymerase chain reaction (RT-PCR) studies showed that aromatase and both estrogen receptors (ERalpha and ERbeta) are expressed in these ganglia. Western blotting also showed that aromatase is expressed in male pelvic ganglia. Using immunohistochemical visualization, ERalpha was predominantly expressed by nitric oxide synthase (NOS)-positive parasympathetic pelvic ganglion neurons. In vivo studies showed that the decrease in pelvic ganglion soma size caused by gonadectomy could be prevented by administration of T or dihydrotestosterone (DHT), but not 17beta-estradiol (E2), showing that this maintenance action of testosterone is mediated entirely by androgenic mechanisms. However, in vitro studies of cultured pelvic ganglion neurons revealed that T, DHT and E each stimulated the growth of longer and more complex neurites in both noradrenergic and cholinergic NOS-expressing neurons. The effects of T were attenuated by either androgen or estrogen receptor antagonists, or by inhibition of aromatase. Together these studies demonstrate that estrogens are likely to be synthesized in the male pelvic ganglia, produced from T by local aromatase. The effects of androgens on axonal growth are likely to be at least partly mediated by estrogenic mechanisms, which may be important for understanding disease-, aging- and injury-induced plasticity in this part of the nervous system.

Transient suppression of the vesicular acetylcholine transporter in urinary bladder pathways following spinal cord injury.

The aim of this study was to examine the expression profile of the vesicular acetylcholine transporter (VAChT), which is a cholinergic pre-synaptic marker, in the lower neural tract following spinal cord injury (SCI) and its effect on coordination of micturition. In adult female Sprague-Dawley rats, SCI was induced by complete transection of the spinal cord at T9. At various time points, 3, 7, 14 and 28 days, after SCI, cystometry was performed on conscious rats. Bladder areflexia was observed during the first week. Twenty-eight days after SCI the rats showed reflex contractions and voiding. The expression of VAChT was examined with immunohistochemistry. The number of VAChT-positive nerve terminals, which were surrounding neuronal soma, was transiently decreased in pelvic ganglion and spinal cord (L1, L2, L6 and S1). In particular VAChT terminals surrounding motor neurons in the ventral horn and autonomic pre-ganglion cells were dramatically decreased from 3 to 14 days after SCI. Similarly, and the number of VAChT-positive fibers in the bladder wall was also decreased. The intensity of VAChT terminals recovered in all above regions in conjunction with recovery of bladder function. These observations indicate that the transient decrease of the VAChT-positive nerve might cause a failure of cholinergic neuronal transmission along the urinary bladder tract after SCI. As the cholinergic system was recovered at least in rat, the functional recovery of neurogenic bladder syndrome in SCI patients may become possible by further understanding the mechanism underlying the recovery of cholinergic system in rat.

Differential expression of glial cell line-derived neurotrophic factor family receptor alpha-2 isoforms in rat urinary bladder and intestine.

Neurturin (NTN) is a member of the glial cell line-derived (GDNF) family of neurotrophic factors, which act via a receptor complex composed of a signal transducing receptor, c-Ret and a glycosylphosphatidylinositol (GPI)-linked ligand binding receptor, GFRalpha. Different members of the GDNF family bind preferentially to one of four different GFRalpha receptors; NTN binds preferentially to the GFRalpha-2 receptor. Recent evidence has shown that three alternatively spliced isoforms of GFRalpha-2 occur in rodent tissues, including the rat brain, myenteric plexus and kidney, and several mouse tissues. Here we have examined the occurrence of GFRalpha-2 isoforms in the adult male rat urinary bladder by RT-PCR, in parallel with samples from the muscularis externa of the rat ileum. In contrast to the ileum, only a single GFRalpha-2 isoform, the smallest isoform, known as GFRalpha-2c, was detected in the rat urinary bladder. This differential expression of GFRalpha-2 transcripts in bladder and intestine may be related to differences in the roles of NTN in the two tissues and its actions on the neurons that innervate them.

New insights in the neuroanatomy of the human adult superior hypogastric plexus and hypogastric nerves.

BACKGROUND: The superior hypogastric plexus (SHP) is an autonomic plexus, located ventrally to the abdominal aorta and its bifurcation, innervating pelvic viscera. It is classically described as being composed of merely sympathetic fibres. However, post-operative complications after surgery damaging the peri-aortic retroperitoneal compartment suggest the existence of parasympathetic fibres. This immunohistochemical study describes the neuroanatomical composition of the human mature SHP. MATERIAL AND METHODS: Eight pre-determined retroperitoneal localizations including the lumbar splanchnic nerves, the SHP and the HN were studied in four human cadavers. Control tissues (white rami, grey rami, vagus nerve, splanchnic nerves, sympathetic ganglia, sympathetic chain and spinal nerve) were collected to verify the results. All tissues were stained with haematoxylin and eosin and antibodies S100, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP) and myelin basic protein (MBP) to identify pre- and postganglionic parasympathetic and sympathetic nerve fibres. RESULTS: All tissues comprising the SHP and hypogastric nerves (HN) showed isolated expression of TH, VIP and MBP, revealing the presence of three types of fibres: postganglionic adrenergic sympathetic fibres marked by TH, unmyelinated VIP-positive fibres and myelinated preganglionic fibres marked by MBP. Analysis of control tissues confirmed that TH, VIP and MBP were well usable to interpret the neurochemical composition of the SHP and HN. CONCLUSION: The human SHP and HN contain sympathetic and most likely postganglionic parasympathetic fibres. The origin of these fibres is still to be elucidated, however surgical damage in the peri-aortic retroperitoneal compartment may cause pelvic organ dysfunction related to both parasympathetic and sympathetic denervation.

The expression of ß3-adrenoceptor and muscarinic type 3 receptor immuno-reactivity in the major pelvic ganglion of the rat.

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. ß3-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by ß3 agonists has questioned the therapeutic site of action of ß3 agonists in the bladder. The present study was done to explore the possibility that ß3-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing ß3-adrenoceptor immuno-reactivity (ß3AR-IR). The only structures found to express ß3AR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed ß3AR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (M3R-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of ß3-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

Improvement of erectile function in diabetic rats by insulin: possible role of the insulin-like growth factor system.

Erectile dysfunction is commonly experienced in men with diabetes mellitus. We report that the intracavernous pressure (ICP) rise in diabetic rats was 55% of the control and returned to normal following insulin (I) or insulin plus free oxygen scavenger (I + S) treatment. Insulin-like growth factor (IGF) binding protein (IGFBP) -3, -4, and -5 messenger RNA (mRNA) levels in the major pelvic ganglia (MPG) of diabetic rats were elevated by 2-fold, 2.6-fold, and 2.5-fold, respectively. Both I and I + S returned IGFBP-4 and 5 mRNA levels to normal, whereas IGFBP-3 gene expression was severely inhibited. IGFBP-2 gene expression was greatly inhibited by diabetes and was unresponsive to treatment. In the penis of diabetic rats, IGFBP-2 and -4 mRNA levels were low, whereas IGFBP-3 mRNA levels were elevated 10-fold. These effects were reversed by I and I + S. I and I + S also corrected the IGFBP-3 expression pattern. IGF-I gene expression in the penis and MPG was not significantly increased (P < 0.05) by diabetes and returned to normal levels following I or I + S treatment. Because IGFs are potent regulatory factors in vascular tone, this newly described activity of insulin may play an important role in the improvement of erectile function seen clinically and in animal models.

Reactions of rat sympathetic neurons to ethanol exposure are age-dependent.

Age-differences in the sensitivity of peripheral sympathetic neurons to chronic ethanol exposure and ethanol withdrawal were studied in male Wistar rats aged 4 months, 12 months, or 24 to 25 months. The superior cervical ganglia (SCG) of the young (4 months) and the 2-year-old rats responded to a 12-day or 4-week ethanol exposure with significantly increased catecholamine turnover, while the ganglia of the middle-aged rats (12 months) showed only a minor increase in the intensity of catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. Extensive neuronal vacuolation was found in the 4 months ethanol-exposed SCG, probably as a reaction of a subpopulation of neurons to increased stimulation. Ethanol-induced neuronal loss was most prominent in the SCG of the oldest age group. Contrary to the marked changes in SCG functional and morphometric parameters, the pelvic sympathetic neurons in the hypogastric ganglion showed no significant changes after ethanol exposure. The pattern of ethanol-induced morphological alterations found in the present study did not provide unambiquous support for either the "accelerated aging" or the "increased vulnerability" concept regarding ethanol-aging interactions in the nervous system.

Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscopic study.

By using both light and electron microscopic immunocytochemical methods, Met5-Enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive structures were detected in the pelvic ganglion of male rats. Denervation studies were carried out to determine the origin of these immunoreactive fibers and the projection of immunoreactive neurons within the pelvic ganglion. MEAGL-like immunoreactivity was found in numerous axon boutons, some small, intensely fluorescent (SIF) cells, and a few principal ganglion neurons. Most of the immunoreactive nerve fibers formed pericellular plexuses surrounding the ganglion cells. In addition, there were a few scattered varicose fibers. These fiber plexuses could be classified into two types: type I (approximately 90% of fibers), which consisted of 80-120 small boutons that synapsed on either the dendrites (80% of cases) or somata (20% of cases) of principal neurons; and type II (approximately 10% of fibers), which consisted of 20-40 larger boutons that formed axodendritic synapses exclusively. After transection of the hypogastric and pelvic nerves, virtually all of the pericellular fiber plexuses disappeared, whereas the scattered varicose fibers remained. According to their ultrastructure, these remaining fibers were considered to arise from SIF cells. Following the injection of Fast Blue into the bladder wall, some of the MEAGL-like immunoreactive principal neurons were retrogradely labeled. The results of this study indicate that there are two origins for the MEAGL-like immunoreactive fibers detected in the pelvic ganglion: most arise from preganglionic neurons in the spinal cord, and a small proportion may originate from intraganglionic MEAGL-like immunoreactive SIF cells or principal neurons. Some MEAGL-like immunoreactive principal neurons may project to the urinary bladder.

Pharmacological comparison of P2X receptors on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons.

Characteristics of P2X receptors on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia have been studied using the whole cell voltage-clamp technique. Fast application of ATP (100 microM) on to isolated neurons voltage clamped at -70 mV induced a slowly desensitising inward current in 96% of the cells tested. Concentration-response curves for ATP yielded EC50 values of 86 microM, 64 microM and 123 microM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, while alpha,beta-methylene ATP was inactive. The response to ATP was antagonised by suramin, Cibacron blue and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The potency of ATP was increased by extracellular acidification and by co-application of micromolar concentrations of Zn2+, while raising pH decreased it. On rat coeliac ganglion neurons, the EC50 values for ATP were 35 microM and 253 microM at pH 6.8 and 8.0, respectively. On mouse coeliac and pelvic ganglion neurons, altering the pH produced comparable changes. In conclusion, our results indicate that, in contrast to the guinea-pig coeliac ganglion, the characteristics of the P2X receptors present on rat coeliac, mouse coeliac and mouse pelvic ganglia are all identical to those present on rat pelvic ganglion, i.e. they are homomeric P2X2 receptors, or heteromultimers with P2X2 being the dominant subunit.