Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.     

Neural apparatus of the wall of the ductus deferens in the dog

While studying the innervation sources of the deferent duct in 10 dogs, 2-3 nerves have been revealed that take their origin at the pelvic neural plexus and approach the duct together with the blood vessels at the pelvic neural plexus and approach the duct together with the blood vessels at the place where it crosses the ureter ("the vascular-neural hilus"). In the experiment performed in 65 dogs the nature of these sources has been revealed. The motor innervation is presented by the nodes of the celiac plexus, of the lumbar and sacral parts of the sympathetic trunk, of the subceliac, gonadal and splanchic pelvic nerves, and the sensitive innervation is multisegmental and is performed by the intravertebral nodes L2-S3. Quantitative investigation of the degenerated neural fibers in the dog deferent duct wall demonstrates that the innervation sources mentioned above participate te a various degree along the course of the organ. In the "hilus" the nerves of the dog deferent duct are divided into the proximal nad distal groups. The proximal group runs towards the prostate and forms a plexus with large loops connected with the neural plexuses of the urinary bladder, the ureter and the prostate. It has small neural nodes. The distally directed nerves run, together with the blood vessels, in the deferent duct towards the epididymis. In the deferent duct wall, adventitila, muscular and mucous neural plexus are found, the cholinergic component prevailing the adrenergic one. The plexuses are somewhat better developed at the beginning of the deferent duct and they are especially pronounced in the ampule. The receptors of the organ's wall are simple, poorly branching.     

Studies of the guinea-pig epididymis

Summary The innervation of the ductuli efferentes and seven zones of the guinea-pig epididymis was investigated using immunohistochemical, histochemical and electron-micro-scopical techniques. Nerve fibers were localized by use of antibodies against substance P (SP-IR), vasoactive intestinal polypeptide (VIP-IR) and dopamine-beta-hydroxylase (DBH-IR). In the ductuli efferentes and all zones of the epididymal duct, SP-IR is consistently observed in the interstitial tissue and perivascular areas. Histochemistry reveals a significant amount of acetylcholinesterase-containing fibers in the interstitial, perivascular and periductal smooth muscles of the ductuli efferentes and zones V, VI and VII. In contrast to the homogeneous distribution of SP-IR within all zones of the epididymis, VIP-IR is seen only in zones VI and VII. Within these zones, VIP-IR is detected in large amounts in the subepithelial and muscular layers as is a sparse number of SP-IR varicosities. DBH-IR is also seen throughout all zones in the interstitial and perivascular regions with a tendency to increase in zones VI and VII. Transmission electron microscopy (TEM) reveals evidence of a cholinergic (agranular vesicles, AGV), adrenergic (small granular vesicles, SGV) and peptidergic (large granular vesicles, LGV) innervation throughout the interstitial connective tissue of the ductuli efferentes and all epididymal zones. Furthermore AGV are localized in the subepithelial layer, and also co-stored with LGV in the muscular layer of zones VI and VII. No nerve profiles were encountered within the epithelium.Correlation of immunohistochemical findings to TEM counterparts as well as their possible functional role are discussed.Supported by the Deutsche Forschungsgemeinschaft     

Cholinergic innervation of the human stomach.

A histochemical method for the acetylcholinesterase activity was used to establish the parasympathetic components of the gastric coats in man. The four gastric layers contain a rich cholinergic innervation. In the mucosa the positive nerve fibers are located around the gastric glands and between the muscles of the muscularis mucosae. In the submucosa rich interconnected nerve fibers, rare large nerve trunks, and scarce ganglia cells show a strong cholinergic reaction. The muscular layer contains the highest density of cholinergic nerve fibers, isolated or in large bundles. Auerbach's plexus has a strong acetylcholinesterase activity in the nerve cell bodies. The subserous layer is very rich in cholinergic nerve fibers, rarely isolated, but interconnected. The vessels of each gastric layer exhibit a rich cholinergic innervation in the adventitia and the outside part of media.     

The bovine tubouterine junction: general innervation pattern and distribution of adrenergic,cholinergic, and peptidergic nerve fibers

The innervation of the bovine tubouterine junction was studied in sexually mature heifers using antisera against various neuronal markers and a modified acetylcholinesterase method. The vast majority of the nerve fibres in the bovine tubouterine junction belongs to the sympathetic nervous system; peptidergic and cholinergic fibers are restricted to characteristic locations. The endosalpinx in the adovarian portion of the terminal tubal segment is poorly innervated. The mucosa of the aduterine portion and of the tubouterine transitinal region proper receives a strikingly dense innervation, which is observed mainly in combination with a strong vascularisation of specialised mucosal structures. In the endometrium, perivascular nerves accompany the ascending spiral arteries but sporadic contacts between nerve fibres and uterine glands are also observed. From the muscular coat the inner longitudinal layer of the terminal tubal segment is more richly supplied by nerve fibres than the intermediate circular and outer longitudinal layers of the tubouterine junction. No changes in the innervation pattern were seen during the different stages of the sexual cycle.     

Morphological and morphometrical characteristics of the esophageal intrinsic nervous system in the golden hamster

Very little is known about esophageal innervation in the hamster. In the present study, we used protein gene product 9.5 (PGP 9.5) to determine immunohistochemically the architectural features of the enteric nervous system in the hamster esophagus. The myenteric plexus consisted of a loose and irregular network of ganglia and interganglionic nerve bundles. The density of the neurons in the myenteric plexus was relatively low (479 +/- 75/cm(2), n = 5), with a preferentially higher density in the upper cervical portion than other parts of the esophagus. Regional differences in the number of PGP 9.5-positive neurons and ganglia were observed. PGP 9.5-immunoreactive fibers in the ganglia often branched, giving rise to expanding nerve endings of laminar morphology resembling intraganglionic laminar endings described in rats and cats. Fine varicose fibers originating from the secondary plexus were occasionally observed near the motor endplates, suggested a dual innervation of the striated muscle. The submucosal plexus was free from ganglionated plexus. A regional difference in the submucosal nervous network was observed. The number of motor endplates in the inner muscle layer was higher than that in the outer muscle layer.     

Analysis of nerve supply pattern in thoracic duct in young and elderly men

BACKGROUND: Analysis of the innervation pattern of the thoracic duct in young and elderly human subjects has been performed. The subdivision of the vessels in cervical and lumbar region were taken in consideration. MeTHODS AND RESULTS: Immunostaining for general nerve fibers with a PGP 9.5 marker disclosed a diffuse innervation of the thoracic duct in young subjects, which was strongly reduced in elderly subjects. In young subjects, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) immunoreactive fibers, markers of noradrenergic postganglionic sympathetic fibers, were frequent; choline acetyltransferase (ChAT) immunoreactive fibers, marker of cholinergic parasympathetic nerve fibers, were also well represented. Therefore, the influence of sympathetic and parasympathetic nerve systems on the thoracic duct can be confirmed. The immunoreactivity of vasoactive intestinal peptide (VIP), a neuropeptide frequently present in cholinergic parasympathetic nerve fibers, was scarcely present. Dopamine-positive fibers were observed in few short nerve fibers. Substance P (SP)-positive fibers were widely distributed in the medial and intimal smooth muscle layers, suggesting their involvement as contractile modulating fibers and sensitive fibers. In elderly subjects, an evident reduction of all specific nerve fibers analyzed was detected, the ChAT-positive fibers being the most affected. CONCLUSIONS: The lymphatic vessel thoracic duct is able to regulate hydrodynamic lymph flow by intrinsic contraction of its smooth muscle layer. Therefore, analysis of the thoracic duct innervation pattern may be important in assessing the regulation of vessel contraction. These findings called attention to the reduction of lymphatic drainage functionality affecting fluid balance in the elderly.     

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.     

Ultrastructure of cholinergic innervation in the cirrhotic liver in guinea pigs. Neurohistochemical and ultrastructural study

Hepatic cirrhosis was induced in guinea pigs by ligation of the common bile duct and innervation of the liver was studied by fluorescence histochemistry (glyoxylic acid method), acetylcholinesterase (AChE) neurohistochemistry (modified Karnovsky and Roots method), and transmission electron microscopy. In control animals the adrenergic terminals showed connections with endothelial cells, hepatocytes and fat-storing cells, but no cholinergic terminals were evident. Cirrhosis was present 6 weeks after the bile duct ligation and marked fibrosis, accompanied by bile duct proliferation, was evident in the portal areas. Numerous AChE-positive nerve fibers traversed the collagenous bundles in the fibrotic areas, and cholinergic terminals formed close contacts with fibroblasts. Each axon terminal was found to contain numerous small coreless vesicles and AChE-reaction products were confirmed in the space between a nerve terminal and a fibroblast. In contrast, fluorescence adrenergic nerve fibers and their terminals remained unchanged. This study demonstrates that parasympathetic cholinergic innervation participates in some stages in the development of hepatic cirrhosis.     

Ectatic blood vessels in port-wine stains lack innervation: possible role in pathogenesis.

The innervation pattern of port-wine stains was investigated using indirect immunohistochemistry with antibodies to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), calcitonin gene-related peptide (CGRP), and neurofilament (NF). The pathologically dilated vessels in the middle and deep dermis were found to have defective innervation with only single or no nerve fibers in their vicinity, while other structures in the skin showed a normal density of fibers. NSE- and PGP-like immunoreactive (-LI) nerve fibers were observed innervating vessels with a normal morphology and other structures in the skin, such as sweat glands and hair follicles, as free nerve endings and in nerve bundles. The nerve bundles were often seen to pass the ectatic vessels without giving off any branches. CGRP-LI nerve fibers were detected running toward epidermis, whereas no fibers were found around the ectatic vessels. NF-LI fibers were seen innervating normal vessels in dermis, while in relation to the dilated vessels, no or only occasional fibers were observed. The lack of innervation may be of importance for the development of the disease as a result of decreased tonus of the vessels and/or a loss of neuronal trophic factors.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Neurohistochemical study of intestinal innervation in experimental diffuse peritonitis

In rats with diffuse peritonitis obtained by intraabdominal administration of a 10% fecal suspension (0.8 ml X 100g) neurohistochemical methods were used to study the adrenergic and cholinergic innervation of muscular membrane of the small and large intestine. It is shown that the disturbance of cholinergic innervation comes along with the intestinal paresis. Adrenergic innervation, represented by separate bundles, is found to be slightly affected.     

Innervation of the bovine naso-labial glands

An investigation was carried out on the innervation of bovine naso-labial glands. Histochemical techniques were used to establish the presence and distribution of cholinergic fibres and peptidergic fibres containing VIP and Substance P were revealed by immunohistochemical methods. Parasympathetic nerve fibres were localized around the secretory units and intralobular excretory ducts. VIPergic nerve fibres, the only peptidergic fibres encountered, were arranged in a similar pattern. Adrenergic fibres were only seen in the periglandular connective tissue around blood vessels of medium size. We are, therefore, of the opinion that the secretory activity is mainly regulated by the parasympathetic nerves and that the VIP acts as a modulator.     

Innervation of the umbilical vessels

Summary Umbilical vessels of guinea-pig fetuses were studied shortly before birth. In all umbilical cords investigated an innervation of the umbilical vessels is lacking. The intrafetal parts of the umbilical vessels on the other hand are richly innervated. A marked difference in the amount of nerve fibres and the pattern of innervation is found between artery and vein. The artery is supplied by a dense nerve plexus which spins around the media and which originates from nerve bundles within the outer adventitial layers. The comparatively scanty innervation of the vein exhibits a more coarsely meshed net pattern. The nerve bundles in the vein exhibit a close affinity to the vasa vasorum.Number and type of the close contacts between the muscle cells are different in the various sections of the umbilical vessels. Similar to the distribution of nerves they are almost absent in the vessels of the umbilical cord, numerously, however, in the intrafetal parts. Contrary to the innervation, the close contacts in the vein are developed more numerously and more broadly than in the corresponding artery.     

Topography and distribution of nerve fibers in the posterior longitudinal ligament of the rat: an immunocytochemical and electron-microscopical study

The distribution and immunocytochemical characterization of nerve fibers and their terminals in the posterior longitudinal ligament of the rat lumbar vertebral column was studied in whole-mount preparations and serial semithin and ultrathin sections. Differences in the localization, distribution pattern and density of peptidergic and catecholaminergic nerve fibers were found in the vertebral and intervertebral regions of the posterior longitudinal ligament. For immunocytochemistry, free floating specimens were incubated with primary antibodies against protein gene product 9.5, substance P, calcitonin gene-related peptide, dopamine-beta-hydroxylase, vasoactive intestinal polypeptide and neuropeptide Y together with the avidin-biotin-peroxidase method. In whole-mount preparations, the neural marker protein gene product 9.5 is immunostained in all unmyelinated nerve fibers in the posterior longitudinal ligament, thus giving a panoramic view of the nerve fiber plexus. The most striking nerve fiber plexus is localized in the intervertebral region. In this region, the posterior longitudinal ligament is rich in capillaries that form a dense plexus within its ventral part and extend to the outer layer of the annulus fibrosus. The peptidergic and catecholaminergic innervation of the posterior longitudinal ligament is discussed in the context of pain syndromes related to the vertebral column and degenerative lumbar spine diseases.     

Cholinergic, nitrergic and peptidergic (Substance P- and CGRP-utilizing) innervation of the horse intestine. A histochemical and immunohistochemical study

The small and large intestine of adult horses were histochemically and immunohistochemically investigated in order to evidence components of the intramural nervous system. The general structural organization of the intramural nervous system was examined by using Nissl-thionin staining as well as the anti-neurofilament 200 (NF200) immunoreaction, which demonstrated the presence of neurons in the submucous as well as myenteric plexuses. The additional presence of subserosal ganglia was shown in the large intestine. Acetylcholinesterase (AChEase) activity was observed in both the submucous and myenteric plexuses. Localization of acetylcholine-utilizing neurons was also evidenced by immunohistochemical reactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). With both histochemistry and immunohistochemistry possible cholinergic nerve fibres were detected in the inner musculature. The two possible cholinergic co-mediators Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) have been investigated by an immunohistochemical approach. CGRP immunoreactivity was detected in roundish nerve cell bodies as well as in nerve fibres of the submucous plexus, whereas SP immunoreactivity was evidenced in nerve fibres of the tunica mucosa, in nerve cell bodies and fibres of the submucous plexus and in nerve fibres of the myenteric plexus. NADPH-diaphorase reactivity, which is linked to the synthesis and release of nitric oxide, was detected in nerve cell bodies and nerve fibres of both the submucous and myenteric plexuses as well as in a subserosal localization of the large intestine. The nitrergic components were confirmed by the anti-NOS (nitric oxide synthase) immunoreaction. Results are compared with those of other mammals and related to the complex intestinal horse physiology and pathophysiology.     

Catecholamine-synthesizing enzymes in the rat stomach

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.