Pelvic Ganglion Research Articles

Src family kinase potentiates the activity of nicotinic acetylcholine receptor in rat autonomic ganglion innervating urinary bladder

Src family kinases (SFKs), one of the tyrosine kinase groups, are primary regulators of signal transductions that control cellular functions such as cell proliferation, differentiation, survival, metabolism, and other important roles of the cell. One of the crucial functions of SFKs is to regulate the activities of various neuronal channels. In this study, we investigated the modulatory action of SFK on nicotinic acetylcholine receptors (nAChRs) expressed in rat major pelvic ganglion (MPG) neurons innervating the urinary bladder. PP1 and PP2 (5 μM), selective Src-kinase inhibitors, attenuated ACh-induced ionic currents and [Ca 2+] i transients in MPG neurons, whereas PP3, an inactive analogue, had no effect. Blocking the tyrosine kinase activity of Src kinase by pp60 c-src inhibitory peptide also reduced the ACh-induced currents. Conversely, sodium orthovanadate (200 μM), a tyrosine phosphatase inhibitor, significantly augmented the ACh-induced currents. In the kinase assay, the activities of SFKs in MPG neurons were also inhibited by PP2, but not by PP3. These data suggests that SFKs may have a facilitative role on the synaptic transmission in rat pelvic autonomic ganglion.

Adipose Tissue‐Derived Stem Cells Secrete CXCL5 Cytokine with Neurotrophic Effects on Cavernous Nerve Regeneration

Previously we reported that paracrine actions likely mediated the therapeutic effects of adipose tissue-derived stem cells (ADSCs) on a rat model of cavernous nerve (CN) injury. To identify potential neurotrophic factors in ADSC's secretion, test the most promising one, and identify the molecular mechanism of its neurotrophic action. Rat major pelvic ganglia (MPG) were cultured in conditioned media of ADSC and penile smooth muscle cells (PSMCs). Cytokine expression in these two media was probed with a cytokine antibody array. CXCL5 cytokine was quantified in these two media by enzyme-linked immunosorbent assay (ELISA). Activation of Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) by CXCL5 was tested in neuroblastoma cell lines BE(2)C and SH-SY5Y as well as in Schwann cell line RT4-D6P2T by Western blot. Involvement of CXCL5 and JAK/STAT in ADSC-conditioned medium's neurotrophic effects was confirmed with anti-CXCL5 antibody and JAK inhibitor AG490, respectively. Neurotrophic effects of ADSC and PSMC-conditioned media were quantified by measuring neurite length in MPG cultures. Secretion of CXCL5 in these two media was quantified by ELISA. Activation of JAK/STAT by CXCL5 was quantified by densitometry on Western blots for STAT1 and STAT3 phosphorylation. MPG neurite length was significantly longer in ADSC than in PSMC-conditioned medium. CXCL5 was secreted eight times higher in ADSC than in PSMC-conditioned medium. Anti-CXCL5 antibody blocked the neurotrophic effects of ADSC-conditioned medium. CXCL5 activated JAK/STAT concentration-dependently from 0 to 50 ng/mL in RT4-D6P2T Schwann cells. At 50 ng/mL, CXCL5 activated JAK/STAT time-dependently, peaking at 45 minutes. AG490 blocked these activities as well as the neurotrophic effects of ADSC-conditioned medium. CXCL5 was secreted by ADSC at a high level, promoted MPG neurite growth, and activated JAK/STAT in Schwann cells. CXCL5 may contribute to ADSC's therapeutic efficacy on CN injury-induced ED.

Semaphorin 3A inhibits growth of adult sympathetic and parasympathetic neurones via distinct cyclic nucleotide signalling pathways

Semaphorin 3A (Sema3A) is an important secreted repulsive guidance factor for many developing neurones. Sema3A continues to be expressed in adulthood, and expression of its receptor, neuropilin-1 (Nrp-1), can be altered by nerve injury. Autonomic neurones innervating the pelvic viscera are particularly susceptible to damage during pelvic surgical procedures, and failure to regenerate or aberrant growth of sympathetic and parasympathetic nerves lead to organ dysfunction. However, it is not known if adult pelvic neurones are potential targets for Sema3A. The effects of Sema3A and activation or inhibition of cyclic nucleotide signalling were assessed in adult rat pelvic ganglion neurones in culture using a growth cone collapse assay. Sema3A caused growth cone collapse in both parasympathetic and sympathetic neurones expressing Nrp-1. However, the effect of Sema3A was mediated by distinct cyclic nucleotide signalling pathways in each neurone type. In parasympathetic neurones, cAMP and downstream activation of protein kinase A were required for growth cone collapse. In sympathetic neurones, cGMP was required for Sema3A-induced collapse; cAMP can also cause collapse but was not required. Sema3A-mediated, cGMP-dependent collapse in sympathetic neurones may require activation of cyclic nucleotide-gated ion channels (CNGCs). We propose that Sema3A is an important guidance factor for adult pelvic autonomic neurones, and that manipulation of their distinct signalling mechanisms could potentially promote functional selective regeneration or attenuate aberrant growth. To our knowledge, this is also the first study to implicate CNGCs in regulating growth cone dynamics of adult neurones.

Immunoperoxidase detection of neuronal antigens in full-thickness whole mount preparations of hollow organs and thick sections of central nervous tissue

Immunofluorescently stained whole mounts have proved useful for defining the innervation of the gut and large blood vessels. Nerves supplying other hollow organs are usually studied in sections, which provide much less information. Aiming to describe the entire innervation of rat uterus, we developed a method for immunoperoxidase staining of full-thickness whole mounts that allowed us to visualize all immunoreactive axons. Uterine horns were dissected out, slit open, stretched, pinned flat and fixed. Entire horns were treated with methanol/peroxide, buffered Triton X-100 and normal serum and then incubated in primary antibodies, biotinylated secondary antibodies and avidin-horseradish peroxidase (HRP), each for at least 3 days. Peroxidase reactions revealed immunoreactivity. Immunostained horns were dehydrated, infiltrated with epoxy resin, mounted on slides under Aclar coverslips and polymerized. We treated bladders, gut, major pelvic ganglia and thick sections of perfused medulla oblongata similarly to assess the applicability of the method. Using this method, we could map the entire uterine innervation provided by axons immunoreactive for a variety of antigens. We could also assess the entire tyrosine hydroxylase-immunoreactive innervation in all layers of bladder, gut and ganglia whole mounts and throughout 300 μm sections of medulla. These observations show that this method for immunoperoxidase staining reliably reveals the complete innervation of full-thickness whole mounts of hollow organs and thick sections of central nervous tissue. The method has several advantages. The resin-embedded tissue does not degrade; the immunostaining is non-fading and permanent and neurochemically defined features can be mapped at large scale without confocal microscopy.

Sources of the porcine testis innervation

This study was carried out on three adult male pigs of the large White Polish breed weighing 110-130 kg each. The animals were anaesthetised and injected with retrograde tracer Fast Blue (FB) into right testis. Three weeks later, the pigs were deeply anaesthetised and perfused transcardially with fixative (4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4). Collected ganglia were cut with freezing microtome into 12-μm-thick sections. The sections were examined under a fluorescent microscope (Zeiss). FB-positive neurones were found in pelvic ganglia (anterior pelvic ganglion) (15.4% of all FB(+) neurones), prevertebral ganglia (caudal mesenteric, testicular, aortico-renal and renal ganglia) (59% of all FB(+) neurones), sympathetic chain ganglia (last four lumbar and first three sacral ganglia) (18.1% of all FB(+) neurones) and dorsal root ganglia (DRG) (first three lumbar and first three sacral ganglia) (7.4% of all FB(+) neurones). The majority of FB-positive nerve cell bodies were observed in ipsilateral ganglia, but they were also found in contralateral ganglia (approximately 85% and 15% respectively). Thus, FB-positive neurones were located in the left prevertebral, sympathetic chain and DRG, but surprisingly, they were absent in left anterior pelvic ganglia.

Mechanisms of reflex bladder activation by pudendal afferents

Activation of pudendal afferents can evoke bladder contraction or relaxation dependent on the frequency of stimulation, but the mechanisms of reflex bladder excitation evoked by pudendal afferent stimulation are unknown. The objective of this study was to determine the contributions of sympathetic and parasympathetic mechanisms to bladder contractions evoked by stimulation of the dorsal nerve of the penis (DNP) in α-chloralose anesthetized adult male cats. Bladder contractions were evoked by DNP stimulation only above a bladder volume threshold equal to 73 ± 12% of the distension-evoked reflex contraction volume threshold. Bilateral hypogastric nerve transection (to eliminate sympathetic innervation of the bladder) or administration of propranolol (a β-adrenergic antagonist) decreased the stimulation-evoked and distension-evoked volume thresholds by -25% to -39%. Neither hypogastric nerve transection nor propranolol affected contraction magnitude, and robust bladder contractions were still evoked by stimulation at volume thresholds below the distension-evoked volume threshold. As well, inhibition of distention-evoked reflex bladder contractions by 10 Hz stimulation of the DNP was preserved following bilateral hypogastric nerve transection. Administration of phentolamine (an α-adrenergic antagonist) increased stimulation-evoked and distension-evoked volume thresholds by 18%, but again, robust contractions were still evoked by stimulation at volumes below the distension-evoked threshold. These results indicate that sympathetic mechanisms contribute to establishing the volume dependence of reflex contractions but are not critical to the excitatory pudendal to bladder reflex. A strong correlation between the magnitude of stimulation-evoked bladder contractions and bladder volume supports that convergence of pelvic afferents and pudendal afferents is responsible for bladder excitation evoked by pudendal afferents. Further, abolition of stimulation-evoked bladder contractions following administration of hexamethonium bromide confirmed that contractions were generated by pelvic efferent activation via the pelvic ganglion. These findings indicate that pudendal afferent stimulation evokes bladder contractions through convergence with pelvic afferents to increase pelvic efferent activity.

Pentoxifylline Promotes Recovery of Erectile Function in a Rat Model of Postprostatectomy Erectile Dysfunction

BackgroundCavernous nerve (CN) injury during radical prostatectomy (RP) causes CN degeneration and secondary penile fibrosis and smooth muscle cell (SMC) apoptosis. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that further inhibits multiple cytokine pathways involved in nerve degeneration, apoptosis, and fibrosis. ObjectivesTo evaluate whether PTX enhances erectile function in a rat model of CN injury. Design, Setting and InterventionsForty male Sprague-Dawley rats underwent CN crush injury and were randomized to oral gavage feeding of phosphate-buffered saline (vehicle) or PTX 25, PTX 50, or PTX 100mg/kg per day. Ten animals underwent sham surgery and received vehicle treatment. Treatment continued for 28 d, followed by a wash-out period of 72h. An additional eight rats underwent resection of the major pelvic ganglion (MPG) for tissue culture and examination of direct effects of PTX on neurite sprouting. MeasurementsIntracavernous pressure recording on CN electrostimulation, immunohistologic examination of the penis and the CN distal to the injury site, and length of neurite sprouts in MPG culture. ResultsDaily oral gavage feeding of PTX resulted in significant improvement of erectile function compared to vehicle treatment in all treated groups. After treatment with PTX 50 and PTX 100mg/kg per day, the expression of neuronal nitric oxide synthase in the dorsal penile nerve was significantly higher than in vehicle-treated rats. Furthermore, PTX treatment prevented collagen deposition and SMC loss in the corpus cavernosum. In the CN, signs of Wallerian degeneration were ameliorated by PTX treatment. MPG culture in medium containing PTX resulted in a significant increase of neurite length. ConclusionsPTX treatment following CN injury in rats improved erectile recovery, enhanced nerve regeneration, and preserved the corpus cavernosum microarchitecture. The clinical availability of this compound merits application in penile rehabilitation studies following RP in the near future.

Erectile Function Restoration After Repair of Excised Cavernous Nerves by Autologous Vein Graft in Rats

Cavernous nerves (CNs) injury is the main cause of erectile dysfunction (ED) following radical prostatectomy. Its restoration remains challenging. To investigate the feasibility of erectile function recovery by autologous vein graft after bilateral CNs being excised in a rat model. A total of 36 adult male Sprague-Dawley rats were randomized into three groups. A 5mm segment of CN was excised bilaterally in group B and C. In group B, a 7-mm segment of autologous saphenous vein was interposed at the defect site bilaterally, with two nerve stumps inserted into the vein lumen. Group C underwent no repair. Group A was accepted a sham operation. 4 months later, apomorphine tests were performed on each rat, followed by injection of 4% fluorogold into bilateral corpus cavernous. 5 days later, after monitoring intracorporal pressure (ICP) changes induced by electrostimulation of CN, rats were sacrificed and their bilateral major pelvic ganglions were obtained for detection of fluorogold, and penile tissues of middle shaft were obtained for detecting nitric oxide synthase-containing nerve fibers in penile dorsal nerves. Erectile function was assessed by apomorphine test and ICP monitoring. CN regeneration was judged by fluoroglod tracing and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. Apomorphine tests resulted in 58% rats with erectile responses in group B, whereas no erection was observed in group C. ICP monitoring also demonstrated a significant recovery in erectile function in group B compared with group C. Much more and brighter fluorogold coloring cells were examined in major pelvic ganglions of group B than those of group C. NADPH-diaphorase staining also showed much more positive fibers were detected in penile dorsal nerves in group B than in group C. Autologous vein graft could provide a guide channel to induce CN regeneration and successfully restore autonomic erectile function after CNs being excised in rats.

Exploring the mechanisms of intravesical electrical stimulation in the in vitro rat whole bladder after treatment with atropine, α,β-methylATP and tetrodotoxin

In a previous study, we showed that the working mechanism of intravesical electrical stimulation (IVES) is probably mainly nerve mediated. But even after bladder decentralization, IVES can induce detrusor contraction. This study explores the effect of IVES in decentralized bladders and the importance of receptors in the bladder wall for a response on IVES. IVES (10 Hz square wave pulses, 20 msec pulse duration, 6 mA) was used in the bladder of 16 female Sprague-Dawley rats. After repeating IVES after consecutive bilateral bladder nerves section (L6-roots, pelvic nerves, and major pelvic ganglion (MPG)), the bladders were mounted in a tissue bath. IVES was performed in the control (n=16), after administration of tetrodotoxin (TTX) (n=6), after atropine and atropine with α,β-methylATP (n=6), and after α,β-methylATP and α,β-methylATP with atropine (n=4). The IVES-induced pressure rise (ΔP) was recorded. Maximum ΔP (maxΔP) after transection of the MPG was significantly lower than after pelvic nerves transection. Treatment with TTX and with α,β-methylATP plus atropine abolished ΔP. Atropine alone gave an insignificant decrease of maxΔP. Treatment with α,β-methylATP alone reduced maxΔP significantly. IVES can evoke contractions in a decentralized bladder. IVES-induced contractions are not a result of direct muscle stimulation, but are nerve mediated, involving intramural innervation and several parts of the bladder innervation. IVES-evoked contraction can be divided in a, contraction duration determining, cholinergic part and a, contraction strength determining, purinergic part. The peripheral innervation could play a role in IVES treatment in patients with interrupted central reflex pathway.

Effect of the artificial somato-autonomic neuroanastomosis on defecation after spinal cord injury and its underlying mechanisms

Objective A new artificial somatic-autonomic neuroanastomosis has been established in male rats with spinal cord injury (SCI). Anorectal manometry and neural retrograde tracing were conducted in such animal model after establishing the regenerated reflex pathway to analyze the mechanisms,and the effects on restoring anorectal function and rectum external sphincter synergetic status. Methods In normal male Sprague-Dawley rats weighing 280-300 g, under anesthesia the rats were subjected to limited laminectomy of L4-L6 vertebrae to expose the spinal cord, and the new reflex pathways were established by intradural microanastomosis of left L4 ventral root to the L6 ventral root while leaving the L4 dorsal root intact to trigger the new defecation reflex pathway. Then L4VR-L6VR formed the foreign nerve root. Three months later the spinal cord was completely transected at the T9-10 level. Eight weeks later the model rats were randomly divided into two groups (n1=8,n2=4). The rats in the group 1 were applied in anorectal manometry, and those in the group 2 for neural retrograde tracing study. Results Stimulation of the L4VR proximal end to the anastomosis and the left sciatic nerve induced rectum to contract, and simultaneously greatly weakened electric activity of external anal sphincter (EAS) ( indicating synergetic relaxation of EAS). But stimulation of the contralateral L6VR evoked simultaneous contraction of rectum and EAS. After FG was injected into the left major pelvic ganglia (MPG) and TMR into the EAS, FG-TMR dual labeled neurons and FG or TMR single labeled neurons were mainly observed in the angulus anterior of L4segment in the model rats. FG-TMR positive neurons were not observed in the spinal cord of the normal rats. Conclusion After establishment of the artificial somato-autonomic neuroanastomosis the regenerated neural pathways can effectively improve the rectum EAS synergetic status and restore the anorectal function.Simultaneous reinnervation of MPG and EAS by one group of the dual labeled neurons in L4 may be the key neural anatomy infrastructure for controllable defecation via the somatic-autonomic reflex pathway. Key words: Somato-autonomic neuroanastomosis; Spinal cord injury; Synergetic defecation

Origin and Characterization of Retrograde Labeled Neurons Supplying the Rat Urethra Using Fiberoptic Confocal Fluorescent Microscopy In Vivo and Immunohistochemistry

Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may increase urethral resistance or regulate the reflex mechanisms controlling continence.

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca 2+ ([Ca 2+] i) and Na + concentrations ([Na +] i) in MPG neurons. The recovery of [Na +] i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na +/K + ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% ( n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca 2+] i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca 2+-activated K + channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na +] i and [Ca 2+] i, which activates Na +/K + ATPase and Ca 2+-activated K + channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.

Immunohistochemical characterization of pelvic neurons projecting to the bladder in rats with the artificial somatic-autonomic reflex arc

Objective Retrograde-tracing and immunohistochemical techniques were used in combination to investigate the types of putative transmitters in pelvic ganglia neurons projecting to the bladder of rats with the artificial somatic-autonomic reflex arc. Methods The artificial somatic-autonomic reflex arc was established in 8 rats by end-to-end microanastomosis of the proximal stump of the left L4 ventral root to the distal stump of L6 ventral root. The major pelvic ganglia (MPG) neurons innervating the urinary bladder were labeled by retrograde axonal tracing methods with the use of horseradish peroxidase (HRP) injected into the bladder wall and identified with TMB-HRP reaction. Then immunohistochemical (ChAT, TH, VIP, SP) and histochemical methods (NADPH-diaphorase activity and nitric oxide synthase immunoreac-tivity) were used to identify the neurotransmitters of HRP-labeled neurons projecting to the bladder. Results HRP-labeled neurons with spherical or oval shape mainly distributed in the MPG. The HRP-labeled neurons were positive for ChAT (112/279, 40. 1%), TH (50/245,20.4%), VIP(25/233, 10.7%), NOS ( 15/230, 6. 5% ) or SP (8/242, 3. 3% ) respectively. Conclusion These results suggest that ace-tylcholine and adrenaline are the predominant neurotransmitters of postganglionic neurons innervating bladder in rats with the artificial somatic-autonomic reflex arc. VIP, NO and SP may be as the neurotransmitters or neuromodulators to participate the procedure of urinary storage and voiding. Key words: Bladder; Pelvic ganglion; Neurotransmitter; Artificial reflex arc

Choline acetyltransferase-and nitric oxide synthase-containing neurons of the major pelvic ganglion projecting to the external urethral sphincter in rats

Objective To observe the choline acetyltransferase (ChAT)-and nitric oxide synthase ( NOS) -containing neurons of the major pelvic ganglion ( MPG) projecting to the external urethral sphincter (EUS). Methods Twenty-four Sprague-Dawley rats were divided into two groups: group A (n = 12) and group B (n = 12). In group A, the pelvic ganglia neurons innervating the EUS were labeled by retrograde axonal tracing methods with the use of horseradish peroxidase (HRP) injected into the EUS and identified with TMB-HRP reaction. Then immunohistochemical (ChAT) and histochemical methods ( NADPH-diaphorase activity and nitric oxide synthase immunoreactivity) were used to identify the neurotransmitters of HRP-labeled neurons projecting to the EUS. In group B, Malt agglutinator binding horseradish peroxidase (WGA-HRP) was injected into the MPG for antegrade tracing. Results In group A, HRP-labeled neurons with spherical or oval shape mainly distributed in the MPG. The HRP-labeled neurons were positve for ChAT (155/358, 43.3%) and NOS (49/339, 14.5%), respectively. In group B, HRP positive axon terminals were observed in the EUS after WGA-HRP was injectec into MPG. Conclusion ChAT and NOS-containing neurons of the major pelvic ganglion project to the ELS. Acetylcholine and nitric oxide may be as neurotransmitters or modulators to participate the procedure of urinary storage and voiding. Key words: Pelvic ganglion; External urethral sphincter; Choline acetyltransferase; Nitric oxide svnthase

VIP and PACAP Effects on Mouse Major Pelvic Ganglia Neurons

Major pelvic ganglia (MPG) neurons innervate urogenital organs and components of the lower bowel. Immunoreactivity for vasoactive intestinal polypeptide (VIP) has previously been observed in the MPG, and VIP knockout animals have impaired micturition reflexes suggesting a role for this neuropeptide in urogenital function. Here, we investigate the presence and action of VIP and a related neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the pelvic ganglia of male mice. An abundance of VIP-immunoreactive (IR) neurons and nerve fibers were observed in the ganglion, whereas PACAP immunoreactivity was not seen. Extracts from acutely isolated MPG contained transcripts for the VPAC1, VPAC2, and PAC1 receptors. Local application of VIP, PACAP, or maxadilan to isolated pelvic ganglion neurons shortened the duration of the afterhyperpolarization (AHP) of action potentials elicited by brief intracellular depolarization. All three peptides also increased neuronal excitability within a subpopulation of the sampled neurons. Bath application of apamin, a peptide antagonist of SK channels, shortened the duration of the AHP indicating that AHP duration in pelvic neurons is determined principally by SK-channel activity. The results suggest that VIP has a role in the neural control of pelvic organ function and activation of VPAC and/or PAC1 receptors can modulate the activity of the autonomic neurons innervating pelvic organs.

PACAP Expression in Explant Cultured Mouse Major Pelvic Ganglia

The major pelvic ganglia (MPG) contain both parasympathetic and sympathetic postganglionic neurons and provide much of the autonomic innervation to urogenital organs and components of the lower bowel. Whereas many parasympathetic neurons were found to express vasoactive intestinal polypeptide (VIP), no MPG neurons exhibited immunoreactivity for pituitary adenylate cyclase-activating polypeptide (PACAP). However, in 3-day cultured MPGs, numerous PACAP-IR cells and nerve fibers were present, and transcript levels for PACAP increased significantly. In 3-day cultured MPGs, PACAP immunoreactivity was seen in cells that were also immunoreactive for VIP or neuronal nitric oxide synthase, but not tyrosine hydroxylase, indicating that PACAP expression occurred preferentially in MPG parasympathetic postganglionic neurons. Transcript levels for the VPAC2, but not VPAC1 or PAC1 receptor, also increased significantly following 3days in culture. Transcript levels of activating transcription factor 3 (ATF-3), a marker of cellular injury, were increased 64-fold in 3-day explants, and ATF-3-IR nuclei were evident in both TH-IR and nNOS-IR neurons as well as in non-neuronal cells. In sum, these results demonstrate that, although only the parasympathetic neurons in explant cultured MPGs increase expression of PACAP, both sympathetic and parasympathetic postganglionic neurons in the cultured MPG whole-mount increase expression of ATF-3.

Erectogenic and Neurotrophic Effects of Icariin, a Purified Extract of Horny Goat Weed (Epimedium spp.) In Vitro and In Vivo

Epimedium species (aka horny goat weed) have been utilized for the treatment of erectile dysfunction in Traditional Chinese Medicine for many years. Icariin (ICA) is the active moiety of Epimedium species. To evaluate the penile hemodynamic and tissue effects of ICA in cavernous nerve injured rats. We also studied the in vitro effects of ICA on cultured pelvic ganglia. Rats were subjected to cavernous nerve injury and subsequently treated for 4 weeks with daily gavage feedings of a placebo solution of normal saline and Dimethyl sulfoxide (DMSO) vs. ICA dissolved in DMSO at doses of 1, 5, and 10 mg/kg. A separate group underwent a single dose of ICA 10 mg/kg 2 hours prior to functional testing. Functional testing with cavernous nerve stimulation and real-time assessment of intracavernous pressure (ICP) was performed at 4 weeks. After functional testing, penile tissue was procured for immunohistochemistry and molecular studies. In separate experiments, pelvic ganglia were excised from healthy rats and cultured in the presence of ICA, sildenafil, or placebo culture media. Ratio of ICP and area under the curve (AUC) to mean arterial pressure (MAP) during cavernous nerve stimulation of subject rodents. We also assayed tissue expression of neuronal nitric oxide synthase (nNOS), eNOS: endothelial nitric oxide synthase (eNOS), calponin, and apoptosis via immunohistochemistry and Western blot. Serum testosterone and luteinizing hormone (LH) were assayed using enzyme-linked immunosorbant assay (ELISA). Differential length of neurite outgrowth was assessed in cultured pelvic ganglia. Rats treated with low-dose ICA demonstrated significantly higher ICP/MAP and AUC/MAP ratios compared with control and single-dose ICA animals. Immunohistochemistry and Western blot were revealing of significantly greater positivity for nNOS and calponin in penile tissues of all rats treated with ICA. ICA led to significantly greater neurite length in cultured specimens of pelvic ganglia. ICA may have neurotrophic effects in addition to known phosphodiesterase type 5 inhibiting effects.

876 CAVERNOUS NERVE REGENERATION BY SONIC HEDGEHOG

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010876 CAVERNOUS NERVE REGENERATION BY SONIC HEDGEHOG Nicholas Angeloni, Christopher Bond, Yi Tang, Shuming Zhang, Daniel Harrington, Kevin McKenna, Samuel Stupp, and Carol Podlasek Nicholas AngeloniNicholas Angeloni Chicago, IL More articles by this author , Christopher BondChristopher Bond Chicago, IL More articles by this author , Yi TangYi Tang Chicago, IL More articles by this author , Shuming ZhangShuming Zhang Chicago, IL More articles by this author , Daniel HarringtonDaniel Harrington Houston, TX More articles by this author , Kevin McKennaKevin McKenna Chicago, IL More articles by this author , Samuel StuppSamuel Stupp Chicago, IL More articles by this author , and Carol PodlasekCarol Podlasek Chicago, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1632AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Apoptosis of penile smooth muscle (SM) caused by cavernous nerve (CN) injury/prostatectomy is a major contributing factor to erectile dysfunction (ED) development. Understanding the mechanisms that regulate CN integrity and regeneration are critical for prevention of penile apoptosis and development of novel therapeutic approaches for ED treatment. Sonic hedgehog (SHH) is a secreted protein that is abundant in pelvic ganglia neurons and Schwann cells of the CN. Schwann cells are a critical factor in promoting axon repair in the milieu of the regenerating peripheral nerve. We propose that SHH in Schwann cells of the CN is essential to maintain CN integrity, and that local SHH protein treatment of the CN at the time of injury will speed CN regeneration and prevent penile apoptosis. In this study we have used novel peptide amphiphile “noodle” nanotechnology for local SHH protein delivery to crushed CNs and examined CN regeneration and penile morphology. METHODS Sprague Dawley rats underwent bilateral CN crush and CNs were treated with SHH noodle for 4 and 6 weeks (n=7, 9). Control rats underwent bilateral CN crush and were treated with BSA/PBS noodle (n=4, 9). Intracavernosal pressure/blood pressure (ICP/BP), electron microscopy (EM), TUNEL, and semi-quantitative immunohistochemical analysis for SHH, growth associated protein 43 (GAP43) and anti-glial fibrillary acidic protein (GFAP) were performed. RESULTS SHH treatment of the CN caused a 58% improvement in ICP/BP (p-value=0.05) 6 weeks after CN injury. SHH treated CNs showed normal morphology, while demyelination and axonal degeneration were abundant in BSA/PBS treated nerves, by EM. GFAP protein increased 22% in BSA/PBS treated CNs (p-value=0.004) while GFAP was indistinguishable from non-surgery rats in the SHH group (p-value=0.1), indicating significant regeneration. Preliminary results suggest a 36% difference in the number of growth cones in SHH and BSA/PBS treated tissues, 6 weeks after injury. CONCLUSIONS These results show that SHH protein delivered by noodle nanotechnology is effective in speeding CN regeneration and has significant clinical potential to be used as a regenerative therapy for the CN in prostatectomy patients. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e343 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nicholas Angeloni Chicago, IL More articles by this author Christopher Bond Chicago, IL More articles by this author Yi Tang Chicago, IL More articles by this author Shuming Zhang Chicago, IL More articles by this author Daniel Harrington Houston, TX More articles by this author Kevin McKenna Chicago, IL More articles by this author Samuel Stupp Chicago, IL More articles by this author Carol Podlasek Chicago, IL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

892 INTERMITTENT CALORIC RESTRICTION MODIFIES NEUROBIOLOGICAL RESPONSE TO BILATERAL CAVERNOUS NERVE CRUSH INJURY IN THE RAT AND FACILITATES RECOVERY OF ERECTILE FUNCTION

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010892 INTERMITTENT CALORIC RESTRICTION MODIFIES NEUROBIOLOGICAL RESPONSE TO BILATERAL CAVERNOUS NERVE CRUSH INJURY IN THE RAT AND FACILITATES RECOVERY OF ERECTILE FUNCTION Robert Payne, Elaine Coderre, Ward T. Plunet, Jeffrey J.L. Carson, Leo P. Renaud, Wolfram Tetzlaff, Rany Shamloul, and Anthony J. Bella Robert PayneRobert Payne Ottawa, Canada More articles by this author , Elaine CoderreElaine Coderre Ottawa, Canada More articles by this author , Ward T. PlunetWard T. Plunet Vancouver, Canada More articles by this author , Jeffrey J.L. CarsonJeffrey J.L. Carson London, Canada More articles by this author , Leo P. RenaudLeo P. Renaud Ottawa, Canada More articles by this author , Wolfram TetzlaffWolfram Tetzlaff Vancouver, Canada More articles by this author , Rany ShamloulRany Shamloul Ottawa, Canada More articles by this author , and Anthony J. BellaAnthony J. Bella Ottawa, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1648AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Preclinical data for therapeutic dietary restriction has been shown to improve outcomes from cervical spinal cord injury. Candidate mechanisms include trkB, the receptor for brain-derived nerve growth factor (BDNF) which is a key cavernous nerve (CN) neuromodulator. The purpose of this study was to determine whether every-other-day-fasting (EODF), a form of intermittent caloric restriction, conferred an erectile recovery advantage following CN crush injury in the rat. METHODS 44 3-month old Sprague-Dawley male rats were used.Controls n=8and cohorts consisting of 9 animals divided into crush-injury only (no EODF), and treatment arms of EODF started 2 weeks prior to injury, EODF started at day of injury, and EODF initiated 2 weeks post-CN injury(EODF until intracavernous pressure measures). ICPchanges standardized to mean arterial pressure (MAP) at 5 months was measured and an extended treatment phase was chosen to wash out short term fasting effects. Tukey-Kramer test was used for post-hoc analysis. Proximal corpora were stained with primary antibodies against tyrosine hydroxylase, neuronal NO synthase (nNOS), and vesicular acetylcholine transporter (VaChT) as described by Nangle and Keast. RESULTS The mean maximal increase in ICP/MAP (with standard deviation) for control animals was 0.673 (.09) versus the crush-control cohort (no caloric modification) ICP/MAP change of 0.17 (0.06). Rats treated with EODF started two weeks prior to injury demonstrated significant (p<0.05) improvement in erectile function with ICP/MAP ratio of 0.37 (0.07) Fasting started day of injury demonstrated less robust recovery, with 0.28 (.05) compared to the “pre-treated” group. The final group, starting EODF two weeks post injury demonstrated results statistically similar to no caloric restriction, with a ICP/MAP ratio of .168 (0.06). The two primary neurobiological endpoints in this study, retrograde axonal transport of fluorogold to the major pelvic ganglion and cavernous body determination of nNOS and VaChT were significantly reduced in crush injury, and 2 week post-injury EODF, compared to EODF initiated 2 weeks prior to/and day of injury. CONCLUSIONS This is the first study to demonstrate endogenous stress-response neuromodulation in a model of radical prostatectomy induced CN injury. Intermittent caloric restriction in the form of EODF confers a recovery advantage for CN function post-injury as measured by ICP/MAP and intact parasympathic neurons. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e348-e349 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Robert Payne Ottawa, Canada More articles by this author Elaine Coderre Ottawa, Canada More articles by this author Ward T. Plunet Vancouver, Canada More articles by this author Jeffrey J.L. Carson London, Canada More articles by this author Leo P. Renaud Ottawa, Canada More articles by this author Wolfram Tetzlaff Vancouver, Canada More articles by this author Rany Shamloul Ottawa, Canada More articles by this author Anthony J. Bella Ottawa, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Professor Thoma Ionescu (1860-1926) founder of the modern Romanian school of surgery.

Professor Thoma Ionescu (1860-1926) founder of the modern Romanian school of surgery.