The Influence of an Adrenergic Antagonist Guanethidine (GUA) on the Distribution Pattern and Chemical Coding of Dorsal Root Ganglia (DRG) Neurons Supplying the Porcine Urinary Bladder.

Although guanethidine (GUA) was used in the past as a drug to suppress hyperactivity of the sympathetic nerve fibers, there are no available data concerning the possible action of this substance on the sensory component of the peripheral nervous system supplying the urinary bladder. Thus, the present study was aimed at disclosing the influence of intravesically instilled GUA on the distribution, relative frequency, and chemical coding of dorsal root ganglion neurons associated with the porcine urinary bladder. The investigated sensory neurons were visualized with a retrograde tracing method using Fast Blue (FB), while their chemical profile was disclosed with single-labeling immunohistochemistry using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase activating polypeptide (PACAP), galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), and calbindin (CB). After GUA treatment, a slight decrease in the number of FB+ neurons containing SP was observed when compared with untreated animals (34.6 ± 6.5% vs. 45.6 ± 1.3%), while the number of retrogradely traced cells immunolabeled for GAL, nNOS, and CB distinctly increased (12.3 ± 1.0% vs. 7.4 ± 0.6%, 11.9 ± 0.6% vs. 5.4 ± 0.5% and 8.6 ± 0.5% vs. 2.7 ± 0.4%, respectively). However, administration of GUA did not change the number of FB+ neurons containing CGRP, PACAP, or SOM. The present study provides evidence that GUA significantly modifies the sensory innervation of the porcine urinary bladder wall and thus may be considered a potential tool for studying the plasticity of this subdivision of the bladder innervation.

Cocaine and Amphetamine Regulated Transcript (CART) Expression Changes in the Stomach Wall Affected by Experimentally Induced Gastric Ulcerations.

Cocaine- and amphetamine-regulated transcript (CART) is a peptide suggested to play a role in gastrointestinal tract tissue reaction to pathology. Gastric ulceration is a common disorder affecting huge number of people, and additionally, it contributes to the loss of pig livestock production. Importantly, ulceration as a focal disruption affecting deeper layers of the stomach wall differs from other gastrointestinal pathologies and should be studied individually. The pig's gastrointestinal tract, due to its many similarities to the human counterpart, provides a valuable experimental model for studying digestive system pathologies. To date, the role of CART in gastric ulceration and the expression of the gene encoding CART in porcine gastrointestinal tube are completely unknown. Therefore, we aimed to verify the changes in the CART expression by Q-PCR (gene encoding CART in the tissue) and double immunofluorescence staining combined with confocal microscopy (CART immunofluorescence in enteric nervous system) in the porcine stomach tissues adjacent to gastric ulcerations. Surprisingly, we found that gastric ulcer caused a significant decrease in the expression of CART-encoding gene and huge reduction in the percentage of CART-immunofluorescent myenteric perikarya and neuronal fibers located within the circular muscle layer. Our results indicate a unique CART-dependent gastric response to ulcer disease.

Paracervical ganglion in the female pig during prenatal development: Morphology and immunohistochemical characteristics.

The present study investigated the development of the paracervical ganglion in 5-, 7- and 10-week-old porcine foetuses using double labelling immunofluorescence method. In 5-week-old foetuses single PGP-positive perikarya were visible only along the mesonephric ducts. They contained DßH or VAChT, and nerve fibres usually were PGP/VAChT-positive. The perikarya were mainly oval. In 7-week-old foetuses, a compact group of PGP-positive neurons (3144±213) was visible on both sides and externally to the uterovaginal canal mesenchyme of paramesonephric ducts. Nerve cell bodies contained only DßH (36.40±1.63%) or VAChT (17.31±1.13%). In the 10-week-old foetuses, the compact group of PGP-positive neurons divided into several large and many small clusters of nerve cells and also became more expanded along the whole uterovaginal canal mesenchyme reaching the initial part of the uterine canal of the paramesonephric duct. The number of neurons located in these neuronal structures increased to 4121±259. Immunohistochemistry revealed that PGP-positive nerve cell bodies contained DßH (40.26±0,73%) and VAChT (30.73±1.34%) and were also immunoreactive for NPY (33.24±1,27%), SOM (23.6±0,44%) or VIP (22.9±1,13%). Other substances studied (GAL, NOS, CGRP, SP) were not determined at this stage of the development. In this study, for the first time, the morphology of PCG formation in the porcine foetus has been described in three stages of development. Dynamic changes in the number of neurons and their sizes were also noted, as well as the changes in immunochistochemical coding of maturing neurons.

Inferior vagal ganglion galaninergic response to gastric ulcers.

Galanin is a neuropeptide widely expressed in central and peripheral nerves and is known to be engaged in neuronal responses to pathological changes. Stomach ulcerations are one of the most common gastrointestinal disorders. Impaired stomach function in peptic ulcer disease suggests changes in autonomic nerve reflexes controlled by the inferior vagal ganglion, resulting in stomach dysfunction. In this paper, changes in the galaninergic response of inferior vagal neurons to gastric ulceration in a pig model of the disease were analyzed based on the authors' previous studies. The study was performed on 24 animals (12 control and 12 experimental). Gastric ulcers were induced by submucosal injections of 40% acetic acid solution into stomach submucosa and bilateral inferior vagal ganglia were collected one week afterwards. The number of galanin-immunoreactive perikarya in each ganglion was counted to determine fold-changes between both groups of animals and Q-PCR was applied to verify the changes in relative expression level of mRNA encoding both galanin and its receptor subtypes: GalR1, GalR2, GalR3. The results revealed a 2.72-fold increase in the number of galanin-immunoreactive perikarya compared with the controls. Q-PCR revealed that all studied genes were expressed in examined ganglia in both groups of animals. Statistical analysis revealed a 4.63-fold increase in galanin and a 1.45-fold increase in GalR3 mRNA as compared with the controls. No differences were observed between the groups for GalR1 or GalR2. The current study confirmed changes in the galaninergic inferior vagal ganglion response to stomach ulcerations and demonstrated, for the first time, the expression of mRNA encoding all galanin receptor subtypes in the porcine inferior vagal ganglia.

Nerve structures of the heart and their immunohistochemical characterization in 10-week-old porcine foetuses.

The aim of the present study was to investigate the distribution of neuronal perikarya and nerve fibres and their chemical coding in the heart of 10-week-old porcine foetuses. The foetuses, obtained from a local slaughterhouse, were fixed by immersion in 4% buffered (pH 7.4) paraformaldehyde. Cryostat sections of the hearts were processed for single- or double-labelling immunofluorescence methods using antibodies against protein gene product (PGP) dopamine beta-hydroxylase (DßH), acetylcholine vesicular transporter (VAChT) and calcitonin gene-related peptide (CGRP). Numerous clusters of the PGP-positive nerve cells were observed throughout the heart wall. The majority of the clusters were found beneath the epicardium around the root of the aorta, pulmonary trunk and main veins. Some single PGP-positive neurons or small clusters of nerve cells were observed in the epicardium of heart atria and ventricles. The richest network of PGP-positive nerve fibres was observed in the base of the heart near the main cardiac blood vessel openings. Many bundles of PGP-positive nerve fibres were observed throughout the four cardiac chambers. Double-immunohistochemical staining revealed that the majority of the neurons contained immunoreactivity to VAChT, some of them stained for CGRP and the single neuronal somata were DßH-positive. The nerve fibres supplying the heart expressed immunoreactivity to DßH, VAChT or CGRP. The distribution and neurochemical coding of the nerve structures in ten-week-old foetuses are different from those observed in the hearts of juvenile pigs.

L'objectif du présent étude était d'examiner la disposition des cellules et des fibres nerveuses, ainsi que de leur codage, aux cœurs de fœtus de porc de 10 semaines. Les fœtus, obtenus dans un abattoir locale, ont été fixés par immersion dans la solution de paraformaldéhyde 4% (pH 7,4). Des coupes de cœurs, obtenues à l'aide du cryostat, ont subi une coloration immunohistochimique simple ou double afin de dépister, dans des structures nerveuses, la présence du produit génétique protéiné (PGP), de la dopamine bêta-hydroxylase (DßH), du transporteur vésiculaire de l'acétylcholine (VAChT) et du peptide relié au gène calcitonine (CGRP). De nombreuses concentrations de cellules nerveuses PGP-positives ont été observées dans la paroi cardiaque. La plupart des concentrations ont été retrouvées en dessous de l'épicarde, autour de la racine de l'aorte, du tronc pulmonaire et des veines principales. Des neurones singulaires PGP-positifs ou des petites concentrations de cellules nerveuses ont été observées dans l'épicarde des oreillettes et des ventricules du cœur. Le plus riche réseau de fibres nerveuses PGP-positives a été observé dans la base du cœur, près de l'acheminement des vaisseaux principaux. Dans les quatre chambres du cœur, de nombreux faisceaux de fibres nerveuses PGP-positives ont été observées. Des colorations immunohistochimiques doubles ont démontré que la plupart de neurones présentaient une immunoréactivité par rapport à VAChT, certains par rapport à CGRP, et des corps singulaires de cellules nerveuses étaient positives par rapport à DßH. Des fibres nerveuses alimentant le cœur présentaient une immunoréactivité par rapport à DßH, VAChT ou CGRP. La distribution et le codage neurochimique des structures nerveuses chez des fœtus de 10 semaines diffèrent de ceux observés dans les cœurs de jeunes porcs.

Innervation of internal female genital organs in the pig during prenatal development.

This study investigated the innervation of internal genital organs in 5-, 7- and 10-week-old female pig foetuses using single and double-labelling immunofluorescence methods. The structure and topography of the organs was examined using a scanning electron microscope (SEM). The investigations revealed differences in the innervation between the three developmental periods. Immunostaining for protein gene product 9.5 (PGP; general neural marker) disclosed solitary nerve fibres in the external part of the gonadal ridge and just outside of the mesenchyme surrounding mesonephric ducts in 5-week-old foetuses. Double-labelling immunohistochemistry revealed that nerve fibres associated with the ridge expressed dopamine ß-hydroxylase (DßH; adrenergic marker) or vesicular acetylcholine transporter (VAChT; cholinergic marker). In 7-week-old foetuses, the PGP-positive nerve terminals were absent from the gonad but some of them ran outside and along, and sometimes penetrated into the mesenchyme surrounding the tubal and uterine segments of the paramesonephric ducts and uterovaginal canal. Few axons penetrated into the mesenchyme. DßH-positive fibres were found in single nerve strands or bundles distributed at the edge of the mesenchyme. VAChT-positive nerve terminals formed delicate bundles located at the edge of the mesenchyme, and the single nerves penetrated into the mesenchyme. DßH was also expressed by neurons which formed cell clusters comprising also DßH- or VAChT-positive nerve fibres. In 10-week-old foetuses, PGP-positive nerve fibres were still absent from the ovary but some were distributed in the mesenchyme associated with the uterovaginal canal and uterine and a tubal segment of the paramesonephric ducts, respectively. DßH- or VAChT-positive nerve fibres were distributed at the periphery of the mesenchyme associated with the uterovaginal canal. Some DßH- and many VAChT-positive nerve fibres were evenly distributed throughout the mesenchyme. The clusters of nerve cells comprised DßH-positive perikarya and DßH- or VAChT-positive nerve fibres. The investigations revealed no DßH/VAChT-positive nerve fibres or neurons as well as no nerve structures stained for calcitonin gene-related peptide and/or substance P (sensory markers) associated with the genital organs in the studied prenatal periods.

Morphological and neuroanatomical study of the mammary gland in the immature and mature European beaver (Castor fiber).

This study investigated general morphology and immunohistochemical properties of nerve fibres supplying the mammary gland (MG) in the European beaver. The microscopic analysis of the beaver mammary gland revealed the presence of morphological structures which are characteristic for mammals. There were no distinct differences in the morphological features of the mammary gland between the juvenile and non-pregnant mature beaver. The nerve fibres were visualized using antibodies against protein gene product 9.5 (PGP) and biologically active substances including ß-hydroxylase tyrosine (DßH), neuropeptide Y (NPY), calcitonine gene-related peptide (CGRP) and substance P (SP). The study has revealed that the MG in the juvenile and mature beaver is richly supplied with PGP-immunoreactive (PGP-IR) nerve fibres. The most abundant innervation was observed in the nipple and less numerous nerve terminals supplied the glandular tissue. Double-labelling immunohistochemistry disclosed that the majority of PGP-IR nerve fibres associated with blood vessels and smooth muscle cells in both the nipple and glandular tissue were also DßH-IR. However, these nerve terminals were less numerous in the glandular tissue than in the nipple. Most of the DßH-IR axons associated with arteries and smooth muscle cells in the entire gland also stained for NPY. Small number of DßH/NPY-IR fibres supplied veins. CGRP-IR fibres were more abundant than those expressing SP. No distinct differences in the distribution and immunohistochemical characteristic of nerve fibres were observed between the juvenile and adult animals. The distribution and immunohistochemical properties of nerve fibres supplying the gland in the beaver remind those previously described in other mammalian species.

The Influence of Gastric Antral Ulcerations on the Expression of Galanin and GalR1, GalR2, GalR3 Receptors in the Pylorus with Regard to Gastric Intrinsic Innervation of the Pyloric Sphincter.

Gastric antrum ulcerations are common disorders occurring in humans and animals. Such localization of ulcers disturbs the gastric emptying process, which is precisely controlled by the pylorus. Galanin (Gal) and its receptors are commonly accepted to participate in the regulation of inflammatory processes and neuronal plasticity. Their role in the regulation of gastrointestinal motility is also widely described. However, there is lack of data considering antral ulcerations in relation to changes in the expression of Gal and GalR1, GalR2, GalR3 receptors in the pyloric wall tissue and galaninergic intramural innervation of the pylorus. Two groups of pigs were used in the study: healthy gilts and gilts with experimentally induced antral ulcers. By double immunocytochemistry percentages of myenteric and submucosal neurons expressing Gal-immunoreactivity were determined in the pyloric wall tissue and in the population of gastric descending neurons supplying the pyloric sphincter (labelled by retrograde Fast Blue neuronal tracer). The percentage of Gal-immunoreactive neurons increased only in the myenteric plexus of the pyloric wall (from 16.14±2.06% in control to 25.5±2.07% in experimental animals), while no significant differences in other neuronal populations were observed between animals of both groups. Real-Time PCR revealed the increased expression of mRNA encoding Gal and GalR1 receptor in the pyloric wall tissue of the experimental animals, while the expression(s) of GalR2 and GalR3 were not significantly changed. The results obtained suggest the involvement of Gal, GalR1 and galaninergic pyloric myenteric neurons in the response of pyloric wall structures to antral ulcerations.

Immunohistochemical characterisation of dorsal root ganglia neurons supplying the porcine mammary gland.

The present study investigated the chemical coding of mammary gland-projecting dorsal root ganglia (DRG) neurons using double-labelling immunohistochemistry. Earlier investigations revealed the presence of Fast blue - positive (FB+) neurons in Th9-Th12 DRG after injection of the tracer into the second, right thoracic mamma. Neurons projecting to the last right abdominal mamma were found in L1-L3 DRG. In the present study, the cryostat sections from these ganglia were stained for calcitonin gene-related peptide (CGRP), substance P (SP), nitric oxide synthase (NOS), galanin (GAL) and pituitary adenylate cyclase activating polypeptide (PACAP). Immunohistochemistry revealed that the vast majority of FB+ mammary gland-projecting neurons contained immunoreactivity to CGRP (68.87±0.7%), SP (63.4±0.9%), NOS (32.47±0.9%), GAL (16.28±0.8%) and less numerous nerve cells stained for PACAP (5.87±0.5%). The present results largely correspond with findings dealing with immunohistochemical characterization of nerve fibres supplying porcine mammary gland structures described earlier.

Vasoactive intestinal polypeptide (VIP)--immunoreactive nerve fibres in the mammary gland of the pig.

Immunohistochemical studies have been performed to investigate the coexistence of VIP with dopamine-beta-hydroxylase (D(beta)H), vesicular acetylcholine transporter (VAChT), somatostatin (SOM) or neuropeptyd Y (NPY) within nerve fibres supplying the immature mammary gland in the pig. Generally, a moderate number of the VIP-immunoreactive (VIP-IR) nerve fibres were located in the nipple and parenchyma of the gland. VIP-IR fibres surrounded smooth muscle cells (SMC), blood vessels (BV) and lactiferous ducts (LD). Double-labelling immunohistochemistry revealed that some of VIP-IR nerve fibres also contained immunoreactivity to D(beta)H. VIP/D(beta)H-IR nerves were associated with BV and SMC and single fibres were observed around the LD in both nipple and parenchyma of the gland. VIP/VAChT-IR nerve fibres were not observed. The majority of VIP-IR fibres associated with SMC were also SOM-IR. Less numerous VIP/SOM-IR fibres supplied the BV and were located around the LD of the gland. A small number of VIP-IR nerves also displayed immunoreactivity to NPY. VIP/NPY-IR nerve fibres supplied the BV of the gland.

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig.

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.

Distribution of ganglionic sympathetic neurons supplying the subcutaneous, perirenal and mesentery fat tissue depots in the pig.

Previous morphological studies revealed that the adipose tissue is innervated by adrenergic nerve fibers. Furthermore, physiological studies showed that the metabolism of adipose tissue is controlled by the adrenergic component of the nervous system. However, nothing is known on the sources of innervation of different fat tissue depots. Therefore, we decided to study the distribution of ganglionic sympathetic neurons innervating adipose tissue in the pig by means of a retrograde tracing method. We used 9 male and 9 female pigs of approximately 50 kg body weight. The retrograde tracer, Fast Blue (FB), was injected into the subcutaneous, perirenal and mesentery fat tissue depots. Results of the present study showed that numerous centers of the sympathetic nervous system innervate adipose tissue in the pig. FB+ neurons projecting to the subcutaneous fat tissue were placed in the thoraco-lumbar region of the sympathetic chain ganglia (SChG). However, neurons supplying perirenal and mesentery fat tissue depots were found in both the SChG and prevertebral ganglia (PVG). We conclude that different adipose tissue depots (subcutaneous, perirenal and mesentery) have different sources of innervation and that there is no significant difference in the distribution of neurons innervating adipose tissue in male and female pigs.