Neural crest-derived neurons invade the ovary but not the testis during mouse gonad development.

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on the genetic sex of gonadal or neural crest cells, and may be blocked by repressive guidance signals elevated in the male pathway. This study reveals another aspect of sexually dimorphic gonad development, establishes a precise timeline and structure of ovarian innervation, and raises many questions for future research.

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations.

Development of testes in the mammalian embryo requires the formation and assembly of several cell types that allow these organs to achieve their roles in male reproduction and endocrine regulation. Testis development is unusual in that several cell types such as Sertoli, Leydig, and spermatogonial cells arise from bipotential precursors present in the precursor tissue, the genital ridge. These cell types do not differentiate independently but depend on signals from Sertoli cells that differentiate under the influence of transcription factors SRY and SOX9. While these steps are becoming better understood, the origins and roles of many testicular cell types and structures-including peritubular myoid cells, the tunica albuginea, the arterial and venous blood vasculature, lymphatic vessels, macrophages, and nerve cells-have remained unclear. This review synthesizes current knowledge of how the architecture of the testis unfolds and highlights the questions that remain to be explored, thus providing a roadmap for future studies that may help illuminate the causes of XY disorders of sex development, infertility, and testicular cancers.

A Randomized, Double-Blind, Controlled Trial Shows that Onabotulinum Toxin A Nerve Blocks do Not Provide Improved Pain Control in Men with Chronic Scrotal Pain.

PURPOSE: The use of onabotulinum toxin A to chemically denervate the testis has been studied as a minimally invasive therapy to treat chronic scrotal pain. To our knowledge no randomized, controlled trials of onabotulinum toxin A for chronic scrotal pain management have been reported to date. MATERIALS AND METHODS: In this double-blind, randomized, controlled trial men with chronic scrotal pain who achieved at least temporary pain relief following a cord block with local anesthesia were randomly assigned to a block using local anesthesia alone vs local anesthesia plus 200 IU onabotulinum toxin A. Standardized assessments of pain levels using a visual analogue score, disease impact, quality of life and mood were performed 1, 2, 3, 4, 12 and 18 weeks after injection. The study primary outcome was the change in the visual analogue score at 1 month. After study completion the men in the control group were given the option to receive onabotulinum toxin A as part of an open label trial. RESULTS: Of 64 men with a mean ± SD age of 45.9 ± 11 years and a mean 5.7 ± 5.7-year history of pain 32 received local anesthesia plus onabotulinum toxin A and 32 received local anesthesia alone. There was no statistically significant difference in any measured outcome when comparing those who received onabotulinum toxin A to controls. Nine of the 13 men (69.2%) in the open label trial achieved an improvement in the visual analogue score (mean group score 6.1 ± 1.66 to 4.5 ± 2.36, Student t-test p=0.022) with a reduction in persistent pain at 3 months in 6 of the 9 (66.7%). CONCLUSIONS: This randomized, double-blind, controlled trial showed no superiority of onabotulinum toxin A plus local anesthesia over local anesthesia alone for pain control in men with chronic scrotal pain. Interestingly, significant pain improvement was noted in our open label onabotulinum toxin A trial, suggesting a potential placebo effect.

Accessory right spermatic ganglion: possible embryological basis and clinical significance.

The spermatic ganglia are collections of sympathetic neuron cell bodies located within the cords of the infrarenal aortic plexus, positioned at the origin of the testicular arteries in males. During routine dissection of the aortic plexus at our institution, one specimen exhibited a second (accessory) testicular artery on the right side that coursed retrocaval. Histology was used to confirm the presence of an accessory right spermatic ganglion at the base of the accessory retrocaval testicular artery. Interestingly, the accessory spermatic ganglion was also supplied by its own right lumbar splanchnic nerve. This is the first case to describe the anatomy of an accessory spermatic ganglion in a specimen that exhibits an accessory testicular artery on the right side. This neurovascular variation is of interest to surgeons who aim to perform nerve-sparing retroperitoneal lymph node dissections for malignancy.

Post-chemotherapy laparoscopic retroperitoneal lymph node dissection is feasible for stage IIA/B non-seminoma germ cell tumors.

OBJECTIVE: To assess the efficacy, outcome and complications of post-chemotherapy laparoscopic retroperitoneal lymph node dissection (L-RPLND) for stage IIA/B testicular germ cell tumor (GCT) patients in comparison with open RPLND (O-RPLND). METHODS: L-RPLND was performed in 14 patients with stage IIA/B non-seminoma GCTs among 154 non-seminoma patients who received RPLND after completion of chemotherapy with tumor marker normalization at our institution between 1998 and 2013. Their outcomes were compared with those of 14 patients with stage IIA/B non-seminoma GCTs treated with O-RPLND during the same period. Clinical parameters were compared between L-RPLND and O-RPLND. RESULTS: There were no significant differences in the background characteristics of the two groups except for follow-up duration (36 months for L-RPLND, 70 months for O-RPLND; p = 0.02). Blood loss during surgery was significantly less for the L-RPLND group than for the O-RPLND group (155 mL for L-RPLND, 700 mL for O-RPLND; p < 0.001). Parameters related to post-operative recovery were significantly better for the L-RPLND group than for the O-RPLND group. Histopathological examination showed no difference between the two groups. Neither group had disease recurrence. CONCLUSION: Post-chemotherapy L-RPLND with a bilateral template and nerve-sparing method was safe, effective, and showed a high preservation rate of antegrade ejaculation with no deterioration of outcomes compared to O-RPLND.

Infertility in rats subjected to genitofemoral nerve section is not associated with testicular damage.

This work was aimed at assessing the relationship between testicular ascent and infertility induced by genitofemoral nerve (GFN) section in rats. Eighteen male rats were assigned to three experimental groups as follows: (i) Group SGFN was subjected to surgical section of genitofemoral nerve; (ii) Group Sham; (iii) Control group. The GFN was cut at puberty (28D), and the contralateral testis removed at 90D, with fertility tests at 120D. At 150D, maturity index, epithelial area and histopathological index of seminiferous tubules of all rats were determined and statistically compared between superior and inferior testicle poles, and between groups. There were no differences in testicular parameters, sperm morphology or sperm concentrations (P > 0.05). Section of NGF interfered with fertility (58.3 ± 15.4 in SGFN versus 83.3 ± 10.5 in Sham) and litter size (6.2 ± 1.1 in SGFN versus 10.7 ± 1.4 in Sham). Cremaster of SGFN group showed early neuropathy. The GFN section induced partial testicular ascent and diminished fertility without damage on testicular morphology or spermatic parameters, because, cremaster could affect the contractibility and ejaculation mechanisms in which it participates. The study of the damage on cremaster induced by an injury on GFN could have an overview of the mechanisms inherent in the testicular ascent induced by this iatrogenic alteration and their potential risks on fertility.

Regression of the mammary branch of the genitofemoral nerve may be necessary for testicular descent in rats.

PURPOSE: Inguinoscrotal testicular descent has been proposed to occur via sensory fibers of the sexually dimorphic genitofemoral nerve, which release a neurotransmitter, calcitonin gene related peptide, to guide the migrating gubernaculum into the scrotum. We hypothesize that androgen mediated regression of the genitofemoral nerve mammary branch is necessary for inguinoscrotal descent in rats. We compared the spatiotemporal development of the genitofemoral nerve in control and antiandrogen treated rats. MATERIALS AND METHODS: A total of 29 Sprague-Dawley® rats were collected (animal ethics committee approval A644) in control and antiandrogen treated groups (flutamide, embryonic days 16 to 19, 75 mg/kg body weight/5% ethanol + oil) on embryonic days 17 and 19, and on postnatal day 2. Sagittal sections of the gubernaculum and its surrounding structures were processed for standard histology and immunohistochemistry for androgen receptor, nerves (Tuj1), calcitonin gene related peptide (marker for genitofemoral nerve) and cell nuclei (DAPI). RESULTS: The inguinal mammary bud, its adjacent androgen receptor and genitofemoral nerve mammary branch (containing calcitonin gene related peptide) persisted from embryonic day 17 to postnatal day 2 in all antiandrogen treated males, yet regressed in all control males by postnatal day 2. CONCLUSIONS: Antiandrogens resulted in the persistence of the mammary branch and inguinal mammary bud. Persistent genitofemoral nerve mammary branches may arrest or slow down gubernacular migration by releasing calcitonin gene related peptide in the mammary inguinal fat pad, thus reducing the chemotactic gradient to calcitonin gene related peptide from genitofemoral nerve branches in the distal scrotum. We hypothesize that this process may be related to antiandrogen induced cryptorchidism in the rodent.

The cholinergic system in rat testis is of non-neuronal origin.

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

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.

Immunohistochemical properties of sympathetic chain ganglia (SChG) neurons projecting to the porcine testis in animals subjected to hemicastration, castration and testosterone supplementation.

The present study was carried out on sexually mature boars. All the animals were injected with Fast Blue into the right testis and then divided into four groups [(group 1--control (G1), group 2--hemicastreatad (G2), group 3 castrated (G3) and group 4--castrated and injected with testosterone (G4) boars)]. After a survival period of 3 weeks, G1 animals were transcardially perfused. In pigs of G2, right testis was removed, whereas in G3 and G4 animals both testes were removed. The pigs of G4 were injected with testosterone. After two weeks, all the animals were transcardially perfused and then their sympathetic chain ganglia were collected. The ganglia were cut into 12 microm-thick cryostat sections. The sections were stained using antisera against TH or DbetaH, VACHT or CHAT, NPY, VIP, GAL and AR. Testosterone plasma levels were evaluated with ELISA test. In control pigs, testosterone level amounted to 8.51 +/- 1.29 ng/ml, whereas in hemicastrated animals it was 1.72 +/- 0.35 ng/ml. Bilateral orchiectomy resulted in a drastic decline in testosterone level. Administration of exogenous hormone resulted in tremendous increase in its plasma level. In control pigs, FB-positive (FB+) neurons were found in the right and left ganglia. Immunohistochemical staining revealed that approximately 74% of FB+ neurons contained immunoreactivity to TH or DbetaH, whereas approximately 4% of FB+ cells were VACHT-positive. Among FB+/DbetaH+ neurons, 72% contained NPY and less than 1% stained for GAL. All FB+/VACHT+ neurons were also VIP+. Approximately 62% of FB+ somata expressed immunoreactivity to NPY, whereas 6% stained for VIP. In all experimental pigs, numbers of FB+ perikarya immunoreactive to TH (from 21% in G3, 26% in G2 to 29% in G4,) and DbetaH (62% in G2, 58% in G3 and 59% in G4,) were smaller than those found in G1 animals, whereas numbers of neurons displaying immunoreactivity to other substances studied were higher. The most significant increases in the number of immunoreactive cells were observed in regard to those expressing GAL (24% in G2, 30% in G3 and 27% in G4,) and VIP (11% in G2, 12% in G3 and 11% in G4,) whereas less distinct changes were observed in case of neurones which stained to NPY (67% in G2, 70% in G3 and 68% in G4,) and VAChT or ChAT (6% in G2, G3 and G4). The most apparent changes in the immunohistochemical features of FB+ neurons evoked by bilateral castration were observed in G3 pigs, whereas changes found in G4 animals were very similar to those observed in G2 pigs.

The spermatic ganglion in humans: an anatomical update.

The male gonad receives nerve fibers from the autonomic ganglionic system. By the present study, we aimed to bring detailed evidences, topographic and structural, on the spermatic ganglia (SG) in humans, as suppliers of autonomic fibers for the testis. We performed retroperitoneal dissections in 25 formalin-fixed human male adult cadavers. Histology used the Hematoxylin-Eosin and we also used Bielschowsky silver stains. Immunohistochemistry used antibodies for tyrosine hydroxylase. In 20÷10 specimens, we identified left spermatic ganglia (LSG) at the aortic origin of the left testicular artery (LTA); in five specimens the LTA left the renal artery but LSG were juxtaposed on the aorta at about the level of origin of a normal LTA. In 15÷25 cadavers, there were right spermatic ganglia (RSG) related to the right testicular artery (RTA) that in 12 cadavers had a precaval disposition. A specimen with retrocaval RTA presented an inferior renal ganglion, supplying both the renal and the RTA. The SG presented renal, lumbar and intermesenteric roots. The inferior branch of the SG connected it to the inferior mesenteric plexus while its infero-lateral branch adjoined the testicular artery. Microscopy confirmed the SG as nervous ganglia and the respective neuronal populations were tyrosine hydroxylase positive, allowing us to consider these ganglia as sympathetic. We bring here the first-time evidence of the SG topography and cathecolaminergic nature in humans; this ganglion may influence the male gonad via the inferior mesenteric plexus and via the vascular path of the testicular artery.

Human cremaster muscle and cremasteric reflex: A comprehensive review.

OBJECTIVE: Human research on the cremaster muscle (CM), cremasteric reflex (CMR) and genitofemoral nerve (GFN) and reports on their clinical applications using electrophysiological and histological techniques are rare. We aimed to present a detailed review of the human CM and CMR based on our earlier publications and relevant literature. METHODS: Electromyography (EMG) of the CM was recorded using disposable needle electrodes. CMR was obtained with tactile and/or electrical stimulation of the inner thigh. Transcranial magnetic stimulation (TMS) and magnetic stimulation of the upper lumbar roots were applied; GFN was stimulated using a surface electrode at the anterior superior iliac spine. RESULTS: CM striated fibers comprised multiple motor end plates. CM needle EMG results were similar to those of the limb muscles in chronic neurogenic disorders. TMS produced clear-cut evoked motor responses from CM. GFN motor conduction time to CM was absent or delayed in patients with inguinal hernia. EMG of CM was abnormal in 40% of patients with premature ejaculation. CONCLUSION: CM is different from other skeletal muscles both morphologically and physiologically. Intersegmental sacrolumbar reflexes are useful for evaluating ejaculatory dysfunction. SIGNIFICANCE: CM is an important muscle for testis thermoregulation and sexual reflexes. Neurophysiological techniques are available for physiological and clinical studies.

Physiopharmacological properties of the testicular capsule: A concise review.

The testicular capsules of different mammalian species exhibit spontaneous motor activity. In addition, contractions can be mediated by neuronal stimulation or exogenous drug administration. However, the physiological role of testicular capsule motor activity is still not well understood. Nevertheless, there is evidence for putative roles in spermatozoa transport from the testis to the caput epididymis, control of interstitial/intratesticular pressure and testicular blood flow. In this review, we have collated information about the agents that regulate testicular capsule motor activity, their receptors and second messengers as well as the impact of altered testicular capsule function on the male reproductive system. Furthermore, we highlight the knowledge gaps in the physiology and pharmacology of the testicular capsule as indicators of future research directions that may lead to a better understanding of the physiological role of testicular capsule motor activity and its importance in male fertility.

Deprival of testicular innervation induces apoptosis of Leydig cells via caspase-8-dependent signaling: a novel survival pathway revealed.

Leydig cells are the primary source of testosterone in adult males. Recently, a growing body of evidence has shown that testicular innervation functions as a major regulator in Leydig cell steroidogenesis. The question then arises whether this novel regulatory pathway also plays an important role in other biological behaviors of this cell type. In the present study, we selectively resected the superior spermatic nerves (SSNs) or the inferior spermatic nerves (ISNs) to investigate the effects of testicular denervation on survival of Leydig cells. After testicular denervation, Leydig cells displayed morphological characteristics of apoptosis, such as chromatin condensation, cell shrinkage and apoptotic body formation. Flow cytometry combined with TUNEL labeling demonstrated dramatic and persistent apoptosis of Leydig cells in the denervated testes 14 and 21 days after operation. Meanwhile, serum T concentrations in the SSN- or ISN-denervated rats dramatically decreased on day 14 and declined further on day 21. Plasma LH levels underwent a remarkable rise, while serum FSH levels remained unchanged. Immunofluorescent staining and flow cytometry further demonstrated that testicular denervation activated caspase-3 and caspase-8, but not caspase-9 in Leydig cells. Our data indicate that testicular innervation functions as an important survival factor for Leydig cells in vivo.

Peptidergic not monoaminergic fibers profusely innervate the young adult human testis.

Numerous studies have reported that intratesticular nerves exert important regulatory effects on the functions of the male gonad; however, as yet little is known about their distribution in the young adult human testis. The purpose of this study was to explore whether peptidergic and adrenergic nerves occur in the male gonad of this age, and, if present, to depict their distribution further. Thirty testes were collected from 15 reproductively healthy donors aged 21-32 years. Antibodies against protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), C-terminal flanking peptide of NPY (CPON) and vasoactive intestinal peptide (VIP) were employed for immunohistochemical detection of intratesticular peptidergic nerves, and those against dopamine-beta-hydroxylase (DBH) and 5-hydroxytryptamine (5-HT) for monoaminergic ones. The testicular parenchyma exhibited a rich innervation by PGP 9.5-positive fibers, mainly associated with Leydig cell nests, blood vessels, and seminiferous tubules. Numerous NPY- and CPON-immunoreactive (IR) nerves also appeared in the gonads, but the vast majority were confined to blood vessels. A small number of VIP-IR fibers were detected in some arterioles. By contrast, however, no fibers displaying DBH or 5-HT immunoreactivity were observed within the testis. Additionally, expression of PGP-9.5, NPY, CPON, VIP, DBH and 5-HT was found in Leydig cells, PGP 9.5 in spermatogonia, and NPY and CPON in peritubular myoid cells. Our results suggest that the young adult human testis is devoid of monoaminergic nerves but profusely innervated by peptidergic fibers, which may serve as major neuronal regulators for testicular functions at this age.

Administration of noradrenaline in the autonomic ganglia modifies the testosterone release from the testis using an ex vivo system.

The male gonad receives nerve fibres from the autonomic ganglionic system. These fibres converge on the testis along two pathways, the superior and the inferior spermatic nerves. The superior spermatic nerve runs from the superior mesenteric ganglion alongside the testicular artery, whereas the inferior spermatic nerve originates in inferior mesenteric ganglion, accompanies the vas deferens and penetrates the inferior pole of the testis. The aim of this work was to evaluate androgen release after the addition of noradrenaline or adrenoreceptor antagonists (propranolol or phentolamine) to the ganglionic compartment. An ex vivo system used in a previous work was incubated in two separate containers, one for the testis and the other for the ganglion. Both organs remain interconnected (as in vivo) by the respective spermatic nerve. When noradrenaline was added to the inferior mesenteric ganglion, testosterone release in the gonad container underwent a progressive and significant increment. Propranolol diminishes and phentolamine increases the androgen release. When using the superior mesenteric ganglion, no changes were observed. These results indicate that the ganglionic stimulation of the autonomic system clearly participates in testosterone release from the testis. This effect depends on the ganglion involved. These results make it evident that not only the classical and well-known hypothalamus-hypophysial axis, but also the peripheral nervous system, via the autonomic ganglia, are directly involved in the endocrine control of the testis.

Pulsed radiofrequency for chronic testicular pain-a preliminary report.

OBJECTIVE: To evaluate the effectiveness of pulsed radiofrequency (PRF) of spermatic cord in the treatment of chronic testicular pain. DESIGN: Ten patients with chronic testicular pain were treated with PRF stimulation of the spermatic cord. A radiofrequency probe placed percutaneously into the spermatic cord was used to deliver four 120-second cycles of 20-millisecond pulses at 2 Hz. Test stimulation was first used to confirm the precise placement of the probe. The short-form McGill Pain Questionnaire was used to assess pain before treatment and at 3 months. Patients who had experienced improvement were followed up by telephone, to determine whether pain relief was sustained. RESULTS: Ten patients were entered into the study but one was lost to follow-up. Of the nine patients evaluated, four had complete resolution of pain, while one had partial pain relief. Three patients experienced no change and one reported that his pain was worse. All patients who experienced complete and partial pain relief continued to do so at a mean long-term follow-up of 9.6 months (range 3-14 months). There were no complications observed immediately or during the follow-up period. CONCLUSION: In this pilot study, pain scores improved in five out of nine patients. PRF of spermatic cord appears to be a safe minimally invasive outpatient procedure that should be investigated further with placebo-controlled trials.

A comparison of high volume/low concentration and low volume/high concentration ropivacaine in caudal analgesia for pediatric orchiopexy.

BACKGROUND: It is unclear whether the volume or concentration of local anesthetic influences its spread and quality of caudal analgesia when the total drug dose is fixed. METHODS: We performed this study in a prospective, randomized, observer-blind manner. Children aged 1-5 yr received a constant dose of 2.25 mg/kg of ropivacaine prepared as either 1.0 mL/kg of 0.225% (low volume/high concentration [LVHC], n = 37) or 1.5 mL/kg of 0.15% solution (high volume/low concentration [HVLC], n = 36). Both solutions contained radiopaque dye. RESULTS: The median spread levels with ranges in the HVLC group (confirmed by fluoroscopic examination) were significantly higher (T6, T3-11) than in the LVHC group (T11, T8-L2). There were no significant differences in recovery times, postoperative pain scores, or side effects between the two groups. After discharge, fewer children in the HVLC group required rescue oral acetaminophen compared with the LVHC group (50.0% vs 75.7%). First oral acetaminophen time was found to be significantly longer with HVLC patients than LVHC patients (363.0 min vs 554.5 min). CONCLUSIONS: We confirmed (with fluoroscopy) that a caudal block with 1 mL/kg ropivacaine spreads to T11 and to T6 with 1.5 mL/kg. If the total dose is fixed, caudal analgesia with a larger volume of diluted ropivacaine (0.15%) provides better quality and longer duration after discharge than a smaller volume of more concentrated ropivacaine (0.225%) in children undergoing day-case orchiopexy. The spread level of ropivacaine correlated significantly with the first oral acetaminophen time after discharge.

Distribution and chemical coding of urinary bladder apex-projecting neurons in aorticorenal and testicular ganglia of the male pig.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in aorticoerenal (ARG) and testicular (TG) ganglia supplying the urinary bladder apex (UBA) in the juvenile male pig (n=4, 12 kg. body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder apex under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG were collected and processed for double-labelling immunofluorescence. The presence of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBA (UBA-PN). The UBA-PN were distributed in both TG and ARG. The majority were found in the left ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBA-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons also contained immunoreactivity to NPY, SOM or GAL. Most of the UBA-PN were supplied with varicose VAChT-, or NOS- IR (immunoreactive) nerve fibres. This study has revealed a relatively large population of differently coded ARG and TG neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

The distribution of nerves supplying the testis, epididymis and accessory sex glands of Suncus murinus.

Chronic testicular pain remains an important challenge for urologists. Investigation of the innervation of male gonads thus becomes essential for deepening our understanding of their regulatory roles in male reproductive physiology and pathophysiology. Studies of testicular innervation are mainly limited to the intratesticular peptidergic nerves of the testis by immunohistochemical and acetylcholinesterase histochemical investigations in some animals. Little is known about the detailed, overall distribution in general experimental animal testis. In this study, the distribution of nerves supplying the testis, epididymis and accessory sex glands of Suncus murinus was investigated by whole mount immunohistochemistry staining using a neurofilament protein antibody. Testicular nerves arose through three routes: nerves deriving from the mesenteric and renal plexuses accompanied the testicular artery, entering into the testicular hilum through the superior ligament of the testis. The nerves originating from the hypogastric plexus then ran along the internal iliac artery, deferential artery, and passed through the mesoductus deferens or mesoepididymis, innervating the cauda and corpus of the epididymis, the vas deferens and the inferior pole of the testis. The third route arose from the pelvic plexus, distributed in the seminal vesicle and the prostate. The density of nerve fibers was higher in the cauda epididymidis than in the testis, and more abundant in the vas deferens. The different origins and distribution densities of testicular nerves in S. murinus may serve different neuronal regulatory functions, and, therefore, S. murinus may be an important model animal for understanding the different characteristics of testicular pain.