Careful Dissection of the Distal Ureter Is Highly Important in Nerve-sparing Radical Pelvic Surgery: A 3D Reconstruction and Immunohistochemical Characterization of the Vesical Plexus.

OBJECTIVE: Radical hysterectomy with pelvic lymphadenectomy (RHL) is the preferred treatment for early-stage cervical cancer. Although oncological outcome is good with regard to recurrence and survival rates, it is well known that RHL might result in postoperative bladder impairments due to autonomic nerve disruption. The pelvic autonomic network has been extensively studied, but the anatomy of nerve fibers branching off the inferior hypogastric plexus to innervate the bladder is less known. Besides, the pathogenesis of bladder dysfunction after RHL is multifactorial but remains unclear. We studied the 3-dimensional anatomy and neuroanatomical composition of the vesical plexus and describe implications for RHL. MATERIALS AND METHODS: Six female adult cadaveric pelvises were macroscopically dissected. Additionally, a series of 10 female fetal pelvises (embryonic age, 10-22 weeks) was studied. Paraffin-embedded blocks were transversely sliced in 8-µm sections. (Immuno) histological analysis was performed with hematoxylin and eosin, azan, and antibodies against S-100 (Schwann cells), tyrosine hydroxylase (postganglionic sympathetic fibers), and vasoactive intestinal peptide (postganglionic parasympathetic fibers). The results were 3-dimensionally visualized. RESULTS: The vesical plexus formed a group of nerve fibers branching off the ventral part of the inferior hypogastric plexus to innervate the bladder. In all adult and fetal specimens, the vesical plexus was closely related to the distal ureter and located in both the superficial and deep layers of the vesicouterine ligament. Efferent nerve fibers belonging to the vesical plexus predominantly expressed tyrosine hydroxylase and little vasoactive intestinal peptide. CONCLUSIONS: The vesical plexus is located in both layers of the vesicouterine ligament and has a very close relationship with the distal ureter. Complete mobilization of the ureter in RHL might cause bladder dysfunction due to sympathetic and parasympathetic denervation. Hence, the distal ureter should be regarded as a risk zone in which the vesical plexus can be damaged.

Ontogeny of sensory and autonomic nerves in the developing mouse skeleton.

BACKGROUND: Bone innervation is implicated in bone modeling and remodeling. This study investigates skeletal nerve development in embryonic and newborn mice, focusing on sensory and autonomic nerves and their temporal occurrence. MATERIALS AND METHODS: The ontogeny of innervation and angiogenesis in the hindlimb skeleton of mice was studied from embryonic day (E) 15 to postnatal day (P) 20. Neuronal tissue was immunohistochemically labeled for detection of growth associated protein 43 (GAP-43), protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY). Vascular endothelium was labeled for platelet endothelium cell adhesion molecule-1 (PECAM-1). Morphology was evaluated with hematoxylin and eosin staining. RESULTS: GAP-43, PGP 9.5, CGRP, and PECAM-1 were all present at E 15, adjacent to areas with high osteogenic and chondrogenic activity. In the primary ossification centers, GAP-43 was found at E 15, PGP 9.5 at E 17, CGRP at E 19, and NPY at P 4. The same time lag in appearance was observed in the secondary ossification centers. The covering capillary network was initially dense, but became mature and sparse from P 12 onwards. CONCLUSION: A functional nerve supply co-localized with a rich capillary network is seen early in the developing mouse skeleton, especially in areas with high osteogenic activity. Sensory innervation occurs prior to partus, while autonomic innervation (revealed by the presence of NPY and TH) is established post partum. The findings indicate a time-related development of nerves with different qualities, according to skeletal development.

Prenatal development of the fetal thoracic sympathetic trunk in sheep (Ovis aries).

This study aims at clarifying the detailed morphological and topographical changes of the thoracic part of the sympathetic trunk of sheep during fetal development. Bilateral micro-dissection of the thoracic sympathetic trunk was performed on 40 sheep fetuses aged 6-20 weeks (18 males and 22 females) under a stereomicroscope. The cervicothoracic ganglion (CTG) was observed on 75/80 sides (93.7%) and was composed of the caudal cervical and the first thoracic ganglia on 45/80 sides (56.2%), and of the caudal cervical and the first two thoracic ganglia on 30/80 sides (37.5%). The presence of the two last (12th-13th) thoracic ganglia was not constant. The influence of the sex, the side of the body, and the ages of the fetus on the morphology and topography of the thoracic sympathetic trunk in sheep were identified. In spite of the differences in the morphology and topography of the thoracic sympathetic trunk between early and late fetal developments, the morphology and topography of the older fetal thoracic sympathetic trunk tended to be similar to that of the adult sheep. To comprehend the comparative morphology of the fetal thoracic sympathetic trunk more completely, our results were compared with previous studies. Consequently, differences and similarities in the composition and position of the CTG, presence of single caudal cervical ganglion without fusion to the thoracic ganglia, and absence of the thoracic ganglia, and presence of splitting of the interganglionic branch were found among sheep, pig, and human fetuses. Therefore, sheep might be the appropriate animal model to be applied in human sympathetic nervous system.

Reconsideration of the autonomic cranial ganglia: an immunohistochemical study of mid-term human fetuses.

The cranial parasympathetic ganglia have been reported to paradoxically contain the sympathetic nerve marker, tyrosine hydroxylase (TH), in addition to neurons expressing parasympathetic markers such as vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS). However, the distribution of these molecules in the cranial ganglia of human fetuses has not yet been examined. Using paraffin sections from 10 mid-term human fetuses (12-15 weeks), we performed immunohistochemistry for TH, VIP, and nNOS in the parasympathetic ciliary, pterygopalatine, otic, and submandibular ganglia, and for comparison, the sensory inferior vagal ganglion. The ciliary and submandibular ganglia contained abundant TH-positive neurons. In the former, TH-positive neurons were much more numerous than nNOS-positive neurons, whereas in the latter, nNOS immunoreactivity was extremely strong. No or a few cells in the pterygopalatine, otic, and inferior vagal ganglia expressed TH. Ciliary TH neurons appeared to compensate for classically described sympathetic fibers arising from the superior cervical ganglion, whereas in the submandibular ganglion, nNOS-positive neurons as well as TH neurons might innervate the lingual artery in addition to the salivary glands. Significant individual variations in the density of all these markers suggested differences in sensitivity to medicine affecting autonomic nerve function. Consequently, in the human cranial autonomic ganglia, it appears that there is no simple dichotomy between sympathetic and parasympathetic function.

Division of autonomic nerves within the neurovascular bundles distally into corpora cavernosa and corpus spongiosum components: immunohistochemical confirmation with three-dimensional reconstruction.

BACKGROUND: Detailed knowledge of the distribution and distal course of periprostatic nerves is essential to improve functional outcomes (erection and continence) after radical prostatectomy (RP). OBJECTIVE: To describe the location of nerve fibres within neurovascular bundles (NVBs) and around the prostate by three-dimensional (3D) computer-assisted anatomic dissection (CAAD) in human foetuses and adult cadavers. DESIGN, SETTING, AND PARTICIPANTS: Serial transverse sections of the pelvic portion were performed in seven human male foetuses and four male adult cadavers. Sections were treated by histologic coloration and neuronal immunolabelling of S100 protein. 3D pelvic reconstruction was achieved with digitised serial sections and WinSurf software. MEASUREMENTS: We evaluated the distribution of nerve fibres within the NVB qualitatively. The distribution of periprostatic nerves was also evaluated quantitatively in the adult specimens. RESULTS AND LIMITATIONS: Periprostatic nerve fibres were dispersed around the prostate on all sides with a significant percentage of these fibres present in the anterior and anterolateral sectors. At the prostate apex and the urethral levels, the NVBs have two divisions: cavernous nerves (CNs) and corpus spongiosum nerves (CSNs). The CNs were a continuation of the anterior and anterolateral fibres around the apex of the prostate, travelling towards the corpora cavernosa. The CSNs were a continuation of the posterolateral NVBs, and they eventually reached the corpus spongiosum. The limitations of this study were the small number of specimens available and the lack of functional information. CONCLUSIONS: The anterolateral position of CNs at the apex of the prostate and the autonomic innervation towards the corpus spongiosum via CSNs indicate possible ways to minimise the effect of prostate surgery on sexual function. The ideal dissection plane should probably include the preservation of the anterolateral tissues and fascias to avoid CN lesions. Anatomic knowledge gained from CAAD pertains directly to proper surgical technique and subsequent recovery of erectile function after RP.

The embryonic brain and development of vagal pathways.

To regulate the autonomic function, the vagus nerve transfers various sensory information from peripheral organs, and appropriate motor reflexes are produced in the neural circuit. The functional development of the vagal pathway during the early phase of embryonic development has long been unclear. Optical recording with voltage-sensitive dyes has provided a new approach to the analysis of the functional development of the embryonic central nervous system. In this review, we present recent progress in optical studies on the vagal pathway in the embryonic chick and rat brainstems. The topics include how neural excitability is initially expressed in the motor and sensory nuclei [e.g. the dorsal motor nucleus of the vagus nerve (DMNV) and the nucleus of the tractus solitarius (NTS)] and how synapse networks are formed in the primary and higher-ordered sensory nuclei [e.g. the parabrachial nucleus (PBN)]. We also refer to the functional development of the glossopharyngeal nuclei and compare the developmental steps with those of the vagal nuclei.

Autonomic innervation of the developing heart: origins and function.

Maintenance of homeostatic circulation in mammals and birds is reliant upon autonomic innervation of the heart. Neural branches of mixed cellular origin and function innervate the heart at the arterial and venous poles as it matures, eventually coupling autonomic output to the cardiac components, including the conduction system. The development of neural identity is controlled by specific networks of genes and growth factors, whereas functional properties are governed by the use of different neurotransmitters. In this review, we summarize briefly the anatomic arrangement of the vertebrate autonomic nervous system and describe, in detail, the innervation of the heart. We discuss the timing of cardiac innervation in the chick and mouse, emphasizing the relationship of the cardiac neural networks to the anatomical structures within the heart. We also discuss the variable contribution of the neural crest to vagal cardiac nerves, and summarize the main neurotransmitters secreted by the developing sympathetic and parasympathetic autonomic divisions. We provide an overview of the main growth factor and gene families involved in neural development, discussing how these factors may impact upon the development of cardiac abnormalities in congenital syndromes associated with autonomic dysfunction.

PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development.

The identification of new signaling pathways critical for cardiac morphogenesis will contribute to our understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. Here, we describe a related signaling pathway involving secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has previously been attributed to abnormalities of neural crest. Here, we show that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis.

Development of AT(1) and AT(2) receptors in the ovine fetal brain.

This study determined the development of AT(1) and AT(2) receptors in the ovine fetal brain from preterm to term by utilizing Western blot for the receptor expression at the protein level, RT-PCR for the receptor mRNA, and immunostaining for the specific receptor immunoreactivity. The results demonstrated that AT(1) and AT(2) receptors developed in an increasing pattern from preterm to term gestational periods in the fetal sheep brain. Both AT(1) and AT(2) receptors have appeared in the major structures in the angiotensin-related central cardiovascular and body fluid controlling pathways at the 0.7 of the gestational age. Importantly, AT(1) receptors have been discovered in the supraoptic nuclei in the fetal hypothalamus, and in the lateral parabrachial nuclei and the ventrolateral medulla in the fetal hindbrain. This provides evidence of the anatomical existence of the angiotensin receptors in the brain areas that are critical for cardiovascular and fluid regulatory functions in utero. In addition, although the results demonstrated the predominance of AT(2) receptors in several regions such as the cerebellum in the ovine fetal brain, dominant occupation of AT(1) receptors in the hypothalamus have appeared early in the life of sheep animals before birth. Together, the data support the hypothesis that the central angiotensin receptors are well developed and established in the last third trimester of gestation. The brain receptors provide a pharmacological basis for the action of angiotensin in the maintenance of in utero fetal physiological functions, including cardiovascular and body fluid balance.

Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish.

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives.

Multipotentiality of the neural crest.

Multiple neural and non-neural cell types arise from the neural crest (NC) in vertebrate embryos. Recent work has provided evidence for multipotent stem cells and intermediate precursors in the early NC cell population as well as in various NC derivatives in embryos and even in adult. Advances have been made towards understanding how cytokines, regulatory genes and cell-cell interactions cooperate to control commitment and differentiation to pigment cells, glia and neurone subtypes. In addition, NC cell fates appeared to be unstable, as differentiated NC cells can reverse to multipotent precursors and transdifferentiate in vitro.

Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members.

How growth factors influence the fate of multipotent progenitor cells is not well understood. Most hematopoietic growth factors act selectively as survival factors, rather than instructively as lineage determination signals. In the neural crest, neuregulin instructively promotes gliogenesis, but how alternative fates are determined is unclear. We demonstrate that bone morphogenic protein 2 (BMP2) induces the basic-helix-loop-helix protein MASH1 and neurogenesis in neural crest stem cells. In vivo, MASH1+ cells are located near sites of BMP2 mRNA expression. Some smooth muscle differentiation is also observed in BMP2. A related factor, transforming growth factor beta1 (TGFbeta1), exclusively promotes smooth muscle differentiation. Like neuregulin, BMP2 and TGFbeta1 act instructively rather than selectively. The neural crest and hematopoietic systems may therefore utilize growth factors in different ways to generate cellular diversity.

Neurohistochemical evaluation of adrenergic innervation in fetal development of the white rat.

Physiological development of adrenergic innervation, an important component of trophic and homeostatic regulation in the mammals was estimated. Our research is directed to evaluate both exo- and endogenous factors contributing to its impairment. The present investigation was carried out on 32 rat fetuses from three various 7-day periods of gestation. Histochemical studies included reactions to catecholamines according to Falck and Owman, and silver staining with AgNO3 according to the Bielschowsky-Gros method. Adrenergic innervation was examined in all fetal body regions, with a special reference to the pelvis. Catecholamines distribution and accumulation in plexuses, ganglia, neurons and chromatophilic cells, as well as serotonine content in mast cells were analyzed. It was found that in the initial period of gestation noradrenaline is absent. In the third week of gestation, the neurotransmitter's accumulation takes place to subsequently decrease gradually until the end of gestation. From this stage synaptic connections of adrenergic innervation with chromatophilic and mast cells could be detected.

Development of nerves containing nitric oxide synthase in the human male urogenital organs.

OBJECTIVE: To determine the spatial and temporal distribution of nitric oxide synthase (NOS) in the urogenital organs of a series of human male fetuses, using an immunohistochemical technique. MATERIAL AND METHODS: Thirteen pre-natal specimens ranging in gestational age from 13 to 30 weeks were acquired following abortion or miscarriage. The distribution of NOS, which catalyses the production of nitric oxide (NO), was revealed using an indirect immunolabelling technique and compared with the overall innervation of each specimen visualized using the general nerve-marker protein gene product 9.5 (PGP). RESULTS: At 13 weeks of gestation the majority of nerves supplying the developing prostate gland expressed NOS while similar nerves formed a very minor proportion of the total innervation to the urinary bladder and intramural ureters. With increasing gestational age, NOS-containing nerves became more numerous in the lower urinary tract, the majority occurring at the bladder neck and around the prostatic urethra. In contrast, NOS-containing nerves were not detected in the muscle coat of the vas deferens and seminal vesicle until 23 weeks of gestation and at 30 weeks still only formed a small proportion of the intramuscular nerves. From 23 weeks onwards NOS-containing nerves were present occasionally in the dense subepithelial nerve plexuses which developed in the bladder, prostate, vas deferens and seminal vesicle. Also from 23 weeks onwards, many of the epithelial cells lining the vas deferens, seminal vesicle and ejaculatory ducts showed immunoreactivity to NOS but no immunoreactivity was observed in the epithelial lining of the urinary bladder and the intramural ureters. CONCLUSION: Based on the comparative density of NOS-containing nerves and the difference in their temporal development among the various urogenital organs it is apparent that NO plays an increasingly important role in the autonomic control of the lower urinary tract during fetal development but that its involvement in the functional control of the vas deferens and seminal vesicle is relatively minor before birth.