The distribution of vesicular acetylcholine transporter in the human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase

Because doubt still remains concerning the distribution of nerves that are unequivocally cholinergic in the human genitourinary organs, we have used a specific marker, namely, an antibody to vesicular acetylcholine transporter (VAChT), to immunolabel cholinergic axons and cell bodies in specimens of urinary bladder, seminal vesicle, vas deferens, and prostate gland obtained from neonates and children post mortem. In addition some sections were double-immunolabeled with VAChT and either neuropeptide Y (NPY) or nitric oxide synthase (NOS). The results demonstrated a rich cholinergic innervation to the muscle coat of the bladder body with a much less prominent, but nonetheless significant, cholinergic innervation to the smooth muscle components of the seminal vesicle, vas deferens, and prostate. Small ganglia were scattered throughout the detrusor muscle of the urinary bladder, approximately 75% of the intramural neurons being VAChT immunoreactive, whereas approximately 95% contained NPY and approximately 40% contained NOS. VAChT immunoreactivity was observed in 40% of neurons in ganglia scattered throughout the pelvic plexus. Almost all these cholinergic neurons contained NPY and approximately 65% contained NOS. Almost all the cholinergic nerve fibers throughout the genitourinary organs also contained NPY. Although NOS was sparse in the cholinergic nerves of the bladder body, it occurred in the majority of cholinergic nerves at the bladder neck and was also present in a proportion of the cholinergic nerves in the other organs examined. VAChT-immunoreactive nerves were also observed in a sub-epithelial location in all the organs examined, the majority containing NPY, whereas a small proportion contained NOS. Although doubt remains about the function of sub-epithelial cholinergic nerves in the urinary bladder, the majority of similar nerves in the seminal vesicle, vas deferens, and prostate gland are considered to be secretomotor. Collectively these findings demonstrate that the cholinergic innervation of the male genitourinary system is well established in the neonate and child. Neurourol. Urodynam. 19:185-194, 2000.     

Immunocytochemical distribution of nitric oxide synthase in the human seminal vesicle: a light and electron microscopical study

Although nitric oxide (NO) has been proven to be one of the most important non-adrenergic, non-cholinergic mediators in the control of human reproductive tract organs, to date information on the significance of NO-mediated signal transduction in the control of human seminal vesicle (SV) function is still sparse. Recent investigations have underlined the significance of NO in the maintenance of sperm capacitation and viscosity of the seminal plasma as well as in the control of mammalian seminal vesicle smooth muscle tone. In order to further investigate the functional impact of NO on the regulation of normal SV function, we examined the distribution of NADPH-diaphorase (NADPH-d), endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the cellular anatomy of human SV by means of light and electron microscopical immunocytochemistry (LM, EM) in combination with the tyramide signal amplification technique. Human SV were obtained from 15 patients who had undergone surgery for pelvic malignancies (carcinoma of the prostate or urinary bladder). SV specimens were fixed, sectioned and examined by LM and EM for the presence of NAPDH-d, eNOS and nNOS using specific antibodies and advanced staining procedures. LM revealed a dense NADPH-d reaction in glandular epithelial structures, whereas no substantial labeling was detected in the fibromuscular stroma. EM showed that the NADPH-d reaction product was abundantly detectable attached to membranes of the endoplasmic reticulum, mitochondria and the nuclei of glandular epithelial cells. nNOS staining was found in nerve fibers branching within the SV tissue. eNOS staining was present in small vessels but was only observed to a minor degree in glandular and subglandular structures and the smooth muscle stroma. Our results support the hypothesis that human SV is a site of NO production. The distribution of NADPH-d may give rise to the speculation that NO is mainly involved in the regulation of SV secretory activity. The sparse correlation between NADPH-d-, eNOS- and nNOS-staining might hint at the existence of a previously unidentified NOS isoform in human SV.     

NADPH-diaphorase in glandular cells and nerves and its relation to acetylcholinesterase-positive nerves in the male reproductive tract of man and guinea-pig

The presence of NADPH-diaphorase activity and acetylcholinesterase in the testis, epididymis, vas deferens, seminal vesicle, pelvic plexus, prostate and urethra of man and guinea-pig was investigated with the nitro blue NADPH technique and the thiocholine method, respectively. In human material NADPH-diaphorase activity was found in the Leydig cells, Sertoli cells and the epithelial linings of the rete testis, the excretory ducts, seminal vesicle, prostate and urethra. The guinea-pig material showed staining of the Leydig cells and spermatozoa and similar epithelial staining of the tract as man. Nerves beneath the epithelium and in the muscle layers of cauda epididymis, vas deferens, seminal vesicle, prostate and urethra were also stained. NADPH-diaphorase-positive nerve cells were seen in the pelvic plexus. Some cells also displayed acetylcholinesterase activity but others showed activity for only one of the enzymes or no activity for either enzyme. In the cauda epididymis, vas deferens, seminal vesicle, prostate and urethra acetylcholinesterase-positive nerve fibres formed a plexus beneath the secretory cells. It is concluded that NADPH-diaphorase, generally accepted as a nitric oxide synthase, is present in glandular cells of the male genital tract. The enzyme is also present in nerves, where it is partly co-localized with acetylcholinesterase. Received: 15 January 1997 / Accepted: 18 September 1997     

Immunohistochemical Localization of Neuropeptide Y in Nerves of the Male Genital Tract of the Photoperiodically Responsive Djungarian Hamster, Phodopus Sungoras/Immunhistochemische Lokalisation von Neuropeptid Y in Nerven der männlichen Genitalorgane des photoperiodisch sensiblen Djungarischen Zwerghamsters, Phodopus sungorus

Neuropeptide Y (NPY) was demonstrated by immunohistochemistry in nerves of the male genital tract of Phodopus sungorus at long (LD 16:8) und short (LD 8:16) photoperiods. No immunoreactive nerve fibres could be demonstrated in the testis, caput and corpus epididymidis and the ventral prostate gland. Dense networks of NPY-containing nerve fibers were demonstrated in the smooth muscle layer of the sperm-transporting duct, beginning in the cauda epididymidis with increasing density towards the distal part of the ductus deferens, and in the smooth muscle layer of the seminal vesicles. At short photoperiods, the density of the NPY-containing nerve plexus decreased only in the smooth muscle layer of the ductus deferens. A "trophic" influence of the large smooth muscle cells of the ductus deferens on their nerves not only in regard to their noradrenaline, but also on their NPY content is discussed.     

Nitroxergic innervation of the human ureterovesical junction

Nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were used to investigate the distribution of nitroxergic, i.e., nitric oxide-synthesizing, neuronal perikarya and processes in the human ureterovesical junction (UVJ). Tissue specimens obtained from two cadaver kidney donors and two patients undergoing radical cystectomy for bladder cancer were examined. Clusters of NOS-immunoreactive neurons were localized in extramural ureterovesical ganglia. NOS-containing nerve fibers traveled within large extramural nerve trunks and marched among smooth muscle bundles. Extramural and intramural blood vessels were encircled by varicose NOS-positive axonal processes. The distribution of NOS immunoreactivity paralleled the staining pattern for NADPH-d activity. Urothelium stained strongly for NADPH-d activity but showed no NOS immunolabeling. Specimens from all four patients investigated showed similar staining patterns. Our results suggest that nitric oxide, a potent smooth-muscle-relaxing neurotransmitter in the autonomic nervous system, plays a physiologic role in opening the human UVJ.     

Nitric Oxide Synthase and Cytochrome C Oxidase Changes in the Tumoural and Peritumoural Cerebral Cortex

Summary. Summary. Background: We analysed changes in nitric oxide synthase (NOS) and cytochrome oxidase (CO) activities in the tumoural and peritumoural cerebral cortex in order to investigate: a) the role of NO in tumourigenesis, in TBF regulation, and in vasogenetic PBE; b) the metabolic changes caused by the neoplasm in the surrounding tissues. Method: Intra-operative samples of cerebral cortex were studied by means of immunohistochemistry for nNOS and iNOS, and by histochemistry for NADPH-diaphorase (NADPH-d) and CO. Findings: In contrast with normal cortex, reactive glial cells and the endothelium of small blood vessels displayed strong NADPH-d and iNOS activities in oedematous peritumoural tissue. In the tumoural cortex, NADPH-d and nNOS-positive neurones were reduced in number and their dendrites were thin and interrupted, and infiltrates of NADPH-d and iNOS-positive tumoural cells were frequent. CO activity was decreased in the deep layers of peritumoural cortex, and it was almost absent in the tumoural cortex. Interpretation: In peritumoural and tumoural cortex changes in NOS and CO activities suggest that the coupling between neuronal activity and blood flow is impaired in the damaged cerebral cortex, and that the increase in NOS activity may play a role in tumour vascularization and progression.     

Robot-assisted laparoscopic radical prostatectomy: an athermal anterior approach to the seminal vesicle dissection

The seminal vesicles, particularly the lateral aspect and tips, are among the closest structures to the cavernous nerves and pelvic plexus. Given this proximity it is essential that the seminal vesicle dissection be performed in an athermal and atraumatic fashion during robot-assisted laparoscopic prostatectomy (RALP). Traditionally the seminal vesicle dissection during RALP is performed by dividing the vas deferens and following it proximally to locate the tip of the seminal vesicle. Here we describe a modification to the traditional anterior approach to seminal vesicle dissection. Our modification allows the dissection to be performed athermally and efficiently with use of minimal traction. The dissection proceeds medially between the two terminal vas deferens to identify the medial surface of one of the seminal vesicles. This medial surface is avascular and can be developed easily along the length of the vesicle using blunt dissection. Once its tip is identified it is elevated with the fourth arm medially between the two vas deferens. The ipsilateral vas can then be clipped and divided below the level of the elevated seminal vesicle. The vascular supply to the seminal vesicle is then simply identified entering the lateral aspect of its tip. A hemolock clip is placed directly beneath the tip of the seminal vesicle to control its vasculature. The remainder of the dissection can be performed with sharp dissection. Using this technique the seminal vesicle can be excised entirely with minimal traction and no thermal energy. By elevating the tip medially away from the location of the pelvic plexus and cavernous nerves, inadvertent damage to these neural structures is avoided when placing the hemolock clips. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

烧伤大鼠小肠肌间神经丛中一氧化氮合酶的组织化学研究

目的　探讨烧伤后大鼠小肠壁内一氧化氮合酶 (NOS)的活性及分布的变化规律。　方法　采用NADPH -黄递酶 (NDP)组织化学法和整装铺片技术对烧伤大鼠小肠壁内NOS的活性及分布进行定量和定位研究。　结果　NOS广泛分布于大鼠小肠壁内 ,主要定位于肌间神经丛。NOS阳性神经元多为圆形和卵圆形 ,神经纤维中多含有膨体 ,形成“串珠”样结构 ,且多与血管和肌纤维伴行。烧伤后肌间神经丛中NOS阳性神经元密度变化不显著 (P >0 .0 5 ) ,但神经元内NOS活性则显著下降(P <0 .0 5～ 0 .0 1) ,同时发现烧伤后NOS阳性神经元结构模糊 ,神经纤维断裂较多 ,未能形成完整的神经纤维网 ,神经纤维中NOS阳性膨体亦明显减少。结论　烧伤后大鼠小肠肌间神经丛中NOS阳性神经结构受损 ,神经元内NOS活性下降 ,同时一氧化氮 (NO)的释放途径亦存在障碍 ,这些变化可能与烧伤后肠道结构受损 ,功能障碍密切相关     

Multiple vasodilator pathways from the pelvic plexus to the penis of the rat.

The main penile or cavernous nerve is usually regarded as the most important vasodilator projection in the rat. Although other descending pathways have been described, there is little detailed information on their importance. In this present report, we provide topographic and quantitative information on lateral and ventral penile branches and examine the vasodilator fibers which join the pudendal neurovascular bundle. Seventeen Sprague-Dawley rats were used. The techniques included injection of dye in the penis to label neurons in the pelvic plexus in combination with transection of the main penile nerve (MPN). NADPH diaphorase (NADPH-d) histochemistry was used to assess the effects of transection of vasodilator pathways on innervation of the penis and for in situ staining of the pelvic plexus. Distinct clusters of penile neurons are aggregated at the origin of several nerve tracts leaving the posterior margin of the major pelvic ganglion (MPG). Multiple NADPH-d+ fiber bundles coursed over the anterior surface of the prostate to reach the penis. Branches from these tracts joined the pudendal neurovascular bundle proximal to the hilum of the penis and provided innervation to the artery throughout its course in the pudendal canal. Consistent with the presence of multiple penile pathways, transection of the MPN reduced, but did not eliminate retrograde labeling of penile neurons in the MPG and only modestly decreased NADPH-d+ fibers in the penis. This study confirms that there are multiple pathways by which vasodilator fibers reach the penis. If a similar allocation of vasodilator output is present in man, preservation of finer branches of the pelvic plexus would be important in surgical procedures on the prostate.     

Nitric oxide synthase in the external urethral sphincter of the sheep: immunohistochemical and functional study

PURPOSE: We studied the distribution of neuronal nitric oxide synthase (nNOS) and the effects of nitric oxide (NO) modulating drugs on contractile function of the external urethral sphincter of lambs. Gender differences were evaluated. MATERIALS AND METHODS: Longitudinal and transverse sections of the external urethral sphincter from 10 female and 10 male lambs were studied using reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and nNOS immunocytochemistry. Isometric contractile responses to electrical field stimulation were recorded from external urethral sphincter preparations from 47 female and 45 male lambs and the effects of NO modulating drugs were evaluated. RESULTS: We detected nNOS in the sarcolemma of some but not all striated fibers, where nNOS seems to be concentrated at the neuromuscular junction. In addition, nNOS was present in nerve fibers and intramural ganglia. The density of innervation decreased toward the distal part of the external urethral sphincter and was higher in male preparations. No significant functional effects of the NOS inhibitor NG-nitro-L-arginine (10 mM.) or the NO donors diethylamine and spermine NONOate (Sigma Chemical Co., St. Louis, Missouri) (5 mM. each) on external urethral sphincter isometric contractility were found in either gender. CONCLUSIONS: Despite the evidence for nNOS at the sarcolemma and nerve fibers of the external urethral sphincter the physiological relevance of these immunohistochemical findings remains to be determined.     

Impaired nitric oxide production of the myenteric plexus in colitis detected by a new bioimaging system

Direct measurement of the release of nitric oxide (NO) from the myenteric plexus has been extremely difficult to date, due to the lack of suitable methodologies. We have developed a new bioimaging system to visualize the nitrergic neurons of the myenteric plexus and investigated whether NO production is impaired in dextran sulfate sodium (DSS)-induced colitis. Longitudinal muscle layers with the myenteric plexus intact were obtained from the rat colon and were incubated with 4,5-diaminofluorescein-2-diacetate (DAF-2DA) (7 microm) for 30 min. Illumination at 450-490 nm revealed the fluorescence in the myenteric plexus. Confocal laser microscopy and three-dimensional reconstruction techniques were used to quantify the changes in the amount of NO production by the myenteric plexus. Fluorescent double-labeled immunostaining for nNOS was performed to confirm the colocalization of nNOS in 4,5-diaminofluorescein (DAF-2)-positive cells. DAF-2 fluorescence was abolished by pretreatment with N(G)-nitro-l-arginine methyl ester (l-NAME; a nonselective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a selective neuronal NOS inhibitor), and omega-conotoxin GVIA (an N-type Ca(2+) channel blocker), but not by nifedipine (an l-type Ca(2+) channel blocker). Fluorescent double-labeled immunostaining showed that DAF-2-positive cells colocalized with nNOS-positive cells. Oral administration of 5% DSS for 7 days induced distal colitis and the number of DAF-2-positive neurons were significantly reduced to 55 +/- 17% of control. DAF-2 offers a sensitive indicator for visualizing production of NO with high spatial resolution. This new system may contribute to the study of the pathophysiological role of the nitrergic pathway in the gastrointestinal tract.     

Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavernosum

AIM: To study the effect of diabetes mellitus and insulin treatment on rat penile nitric oxide synthase content. METHODS: Male Wistar rats were divided at random into two groups: the Control (n = 8) and the Diabetic (n = 17). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. The diabetic animals were then randomly divided into two subgroups: diabetic rats without insulin treatment (n = 7) and diabetic rats with insulin treatment (n = 10). The neuronal nitric oxide synthase (nNOS) in the penile corpus cavernosum were assayed by immumohistochemical staining with specific antibody to nNOS and the nNOS-positive nerve fibers were counted semiquantitatively under a high power microscope. RESULTS: The nNOS- positive nerve fibres in diabetic rats with treatment was higher than that in diabetic rats without treatment (P < 0.05) and lower than that in the controls (P < 0. 01). The nNOS-positive nerve fibres in diabetic rat without treatment were also lower than that in the controls (P < 0. 01). CONCLUSION: In streptozotocin-induced diabetic rats, the nNOS content in the penile corpus cavernosum was significantly decreased. Insulin treatment at the dose level employed partially restores the penile nNOS content in these rats.     

Unilateral renal agenesis with absent ductus deferens, epididymis and seminal vesicle: incidental finding in a 22-year-old patient with maldevelopment of the mesonephric duct

Unilateral renal agenesis with an absence of the seminal vesicle, epididymis and ductus deferens is rare and is the result of a developmental disorder of the mesonephric or Wolffian duct. We report the case of a 22-year-old man who presented with testicular pain on the left side of 3 weeks' duration. During the clinical investigation of the scrotum a nonpalpable ductus deferens on the left side was found incidentally. As a result of the urological ultrasound the diagnosis of renal, epididymal, seminal vesicle and ductus deferens agenesis on the left was confirmed. As a vascular variety the CT demonstrated 2 renal veins and 2 renal arteries on the right originating from the superior mesenteric artery together with the right hepatic artery. The testicular artery was placed on both sides. Further diagnostic investigations including a spermiogram, hormone analysis and kidney function tests were normal. Congenital urogenital malformations can be found in various combinations even in adults. Unilateral absence of the vas deferens during clinical examination should alert the clinician to an underlying renal, seminal vesicle and epididymal anomaly; further urological investigation is mandatory. A genetic investigation of the CFTR gene is not necessary in the absence of both ductus deferentes with renal agenesis.     

人体前列腺外侧神经血管束显微解剖研究

目的了解人体前列腺外侧神经血管束的具体走行和分布。方法采用手术显微镜，对成年男性尸体前列腺外侧神经血管束进行解剖观察，同时采用组织切片神经性一氧化氮合酶(nNOS)免疫组织化学染色方法，对1具成年男尸标本前列腺外侧神经血管束进行染色分析。结果盆丛发出分支与血管一起构成神经血管束，分成两支沿前列腺后外侧和前外侧走行到达尿生殖膈。前列腺后外侧、前外侧神经血管束与尿生殖膈组成三角区，三角区中央可见前列腺包膜，该区无神经血管覆盖。后外侧和前外侧神经血管束中的神经穿过尿生殖膈上筋膜后，在截石位膜部尿道外会合成一支。前列腺外侧神经血管束nNOS免疫组织化学染色，前列腺后外侧和前外侧神经血管束中均存在大量nNOS神经元细胞体和神经纤维。结论前列腺外侧存在2条神经血管束，分别为前外侧和后外侧神经血管束，包含nNOS染色阳性神经节细胞。     

Agenesis of seminal vesicles in infertile males: ultrasonic diagnosis.

We present the incidence of seminal vesicle agenesis and its association with deferens ductus agenesis in 141 males presenting with infertility. Transrectal ultrasonography was performed in every patient. Ten presented seminal vesicle agenesis (8 unilateral and 2 bilateral). Three had absence of the vas deferens on physical examination (3 of 7 with unilateral seminal vesicle agenesis and 1 of 2 with bilateral). Computed tomography confirmed the ultrasound findings. We emphasize the importance of the anomalies as a cause of infertility and the association with other genitourinary anomalies. Moreover, we state the necessity of transrectal ultrasound in the diagnosis.     

Neuroanatomy of the human female lower urogenital tract

PURPOSE: The neuroanatomy of the female lower urogenital tract remains controversial. We defined the topographical anatomy and differential immunohistochemical characteristics of the dorsal nerve of the clitoris, the cavernous nerve and the nerves innervating the female urethral sphincter complex. MATERIALS AND METHODS: A total of 16 normal female human pelvic specimens at 14 to 34 weeks of gestation were studied by immunohistochemical techniques. Serial sections were stained with antibodies raised against the neuronal markers S-100 and neuronal nitric oxide synthase (nNOS), vesicular acetylcholine transporter, calcitonin gene-related peptide and substance P. The serial sections were computer reconstructed into 3-dimensional images. RESULTS: Under the pubic arch at the hilum of the clitoral bodies the branches of the cavernous nerves joined the clitoral dorsal nerve to transform its immunoreactivity to nNOS positive. The cavernous nerves originated from the vaginal nervous plexus occupying the 2 and 10 o'clock positions on the anterolateral vagina and they traveled at the 5 and 7 o'clock positions along the urethra. The urethral sphincter complex was innervated by nNOS immunoreactive and nonimmunoreactive nerve fibers arising from the vaginal nervous plexus and pudendal nerve, respectively. CONCLUSIONS: The dorsal nerve of the clitoris receives nNOS positive branches from the cavernous nerve as a possible redundant mechanism for clitoral erectile function. The urethral sphincter complex has dual innervation, which pierces into the urethral sphincter complex at different locations. The study of the neuroanatomy of the female lower urogenital tract is germane to the strategic design of female reconstructive surgery.     

Nerve injury-related erectile dysfunction following nerve-sparing radical prostatectomy: A novel experimental dissection model

Objectives:   To establish a new experimental rat model in order to define the mechanisms of erectile dysfunction (ED) and to evaluate the changes of neuronal nitric oxide synthase (nNOS) in the pelvic ganglia following nerve-sparing radical prostatectomy.
Methods:   Sprague-Dawley rats were randomized to sham operation, bilateral cavernous nerve dissection (BCND) and bilateral cavernous nerve resection (BCNR) groups. In the BCND group, the cavernous nerves were only dissected bilaterally from the major pelvic ganglion (MPG) to the apex of the prostate without crushing or cutting. At 1, 2, 4 and 8 weeks after surgery, we examined intracavernous pressure along with arterial pressure (ICP/AP), retrograde dye tracing using Fluorogold (FG) and expression of nNOS in the MPG.
Results:   Intracavernous pressure and arterial pressure in the BCND group was significantly decreased at 2 and 4 weeks after surgery compared with the sham group, and improved at 8 weeks. The number of FG-positive cells in the MPG also recovered at 8 weeks. ICP/AP and FG-positive cells in the BCNR group were greatly decreased until 8 weeks. The percentage of nNOS-positive cells per total cells was not different between the sham and BCND groups during the experimental period, whereas that in the BCNR group gradually decreased with time.
Conclusions:   We established a novel rat model, in which cavernous nerve dissection alone caused nerve injury-related ED. We believe that this cavernous nerve dissection model might help clarify the mechanism of nerve injury-related ED and the recovery from ED after nerve-sparing radical prostatectomy.     

男性盆丛的局部解剖特点及其对膀胱前列腺根治性切除的意义

目的:了解男性盆丛神经(IHP)及其分支的局部解剖特点,减少膀胱前列腺根治性切除术中对IHP的医源性损伤。方法:通过解剖12具成年男性尸体骨盆标本,了解IHP及其分支的分布特点。结果:IHP位于腹膜后精囊腺的后外侧,是均一排列的矩形神经丛;IHP纤维分为上、中、下支,上支支配膀胱,中支支配前列腺和精囊,下支纤维支配直肠下部;精囊腺的各面均有IHP纤维支配,以后外侧面为主。IHP的纤维位于膀胱颈的侧面,支配膀胱的侧后壁,膀胱的前表面没有神经支配;IHP与血管形成VAN结构,静脉位于最侧面。海绵体神经起源于IHP的前下方,于前列腺的后外侧形成宽约6mm的神经血管束,在血管的外侧向前下方走行。结论:精囊可作为术中重要的解剖标志,沿精囊和IHP之间的解剖平面分离,可避免对IHP的损伤;紧靠前列腺被膜向下分离至前列腺尖部,并充分显露尿道,可有效地保护海绵体神经血管束。