The Distribution and Chemical Coding of Intramural Nerve Structures Supplying the Porcine Stomach

The present study investigated the arrangement and chemical coding of intramural nerve structures supplying the porcine stomach. Tissue samples comprising all layers of the wall of the ventricular fundus were collected from juvenile female pigs (n = 4), which were first deeply anaesthetized and then transcardially perfused with buffered paraformaldehyde. The cryostat sections were processed for double‐labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene‐product 9.5) and their neurochemical characteristics using antibodies against vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal‐polypeptide (VIP), somatostatin (SOM) and substance P (SP). The study confirmed the presence of three distinct nerve plexuses within the wall of the porcine stomach including one myenteric plexus and two, outer and inner, submucous plexuses. The outer and inner submucous plexuses (OSP and ISP, respectively) were similar in respect to the chemical coding of neurons they contained. Most of the neurons expressed immunoreactivity to SP (ISP 58%; OSP 60%) or to VAChT (ISP 56%; OSP 56%), some of them stained for GAL (ISP 18%; OSP 15%) and solitary nerve cells were SOM‐positive (in ISP only). No neurons in the submucous plexuses displayed immunoreactivity to VIP or NOS. In the myenteric plexus, some neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (8%) or SP (8%) while no neurons immunoreactive for SOM were encountered. In both submucous and myenteric plexuses, many varicose nerve fibres expressed immunoreactivity to VAChT, GAL or SP, while VIP‐, SOM‐ or NOS‐positive nerve terminals were less numerous. The comparison of the present results with those obtained by other authors has revealed distinct inter‐species differences regarding the arrangement and chemical coding of nerve structures supplying the mammalian stomach.     

The distribution and chemical coding of intramural neurons supplying the porcine stomach - the study on normal pigs and on animals suffering from swine dysentery

The present study was designed to investigate the expression of biologically active substances by intramural neurons supplying the stomach in normal (control) pigs and in pigs suffering from dysentery. Eight juvenile female pigs were used. Both dysenteric (n = 4; inoculated with Brachyspira hyodysenteriae) and control (n = 4) animals were deeply anaesthetized, transcardially perfused with buffered paraformalehyde, and tissue samples comprising all layers of the wall of the ventricular fundus were collected. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against vesicular acetylcholine (ACh) transporter (VAChT), nitric oxide synthase (NOS), galanin (GAL), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), Leu(5)-enkephalin (LENK), substance P (SP) and calcitonin gene-related peptide (CGRP). In both inner and outer submucosal plexuses of the control pigs, the majority of neurons were SP (55% and 58%, respectively)- or VAChT (54%)-positive. Many neurons stained also for CGRP (43 and 45%) or GAL (20% and 18%) and solitary perikarya were NOS-, SOM- or VIP-positive. The myenteric plexus neurons stained for NOS (20%), VAChT (15%), GAL (10%), VIP (7%), SP (6%) or CGRP (solitary neurons), but they were SOM-negative. No intramural neurons immunoreactive to LENK were found. The most remarkable difference in the chemical coding of enteric neurons between the control and dysenteric pigs was a very increased number of GAL- and VAChT-positive nerve cells (up to 61% and 85%, respectively) in submucosal plexuses of the infected animals. The present results suggest that GAL and ACh have a specific role in local neural circuits of the inflamed porcine stomach in the course of swine dysentery.     

The influence of experimental ileitis on the neuropeptide coding of enteric neurons in the pig

In the present study, both the ELISA test and immunohistochemical staining were used to investigate the influence of artificially induced ileitis on the chemical coding of enteric neurons in the pig. The ileum wall in experimental (E) pigs was injected in multiple sites with 4% paraformaldehyde to induce inflammation, while in the control (C) animals, the organ was injected with 0.1M phosphate buffer (pH 7.4). Three days after ileitis induction, samples of ileum wall from all the animals were evaluated for VIP, SP, CGRP, NPY, GAL and SOM concentration (ELISA test) and the expression of these biologically active substances by the enteric neurons (immunohistochemical staining). Quantitative results showed that ileitis decreased tissue concentration of VIP, CGRP and SOM but increased tissue concentration of SP, NPY and GAL. Immunochemistry revealed that in both the experimental and control pigs, VIP-positive (VIP+) nerve fibers supplied mainly ileal blood vessels, and the labeled pericarya were located in the inner (ISP) and outer submucous plexus (OSP). SP+ and CGRP+ nerve terminals were found in both the mucous and muscular membrane, while the labeled pericarya were found in ISP, OSP and myenteric plexus (MP). In both C and E pigs, the very few nerve terminals containing NPY and SOM were located mainly in the mucous membrane. NPY- or/and SOM-immunopositive nerve cell bodies were found in ISP, OSP and MP. GAL+ nerve fibers supplied all layers of the ileum and were most numerous in the muscular membrane, while the labeled pericarya were present in all the enteric plexuses. The present results suggest that enteric neurons are highly plastic in their response to inflammation.     

GAL‐ and VIP‐Immunoreactive Nerve Structures in the Ileum and Large Intestine of Pigs with Dysentery

The morphology, neurochemistry and function of intramural nerve structures in the mammalian gastrointestinal tract are relatively well known, but in normal, healthy individuals. The present study was aimed at investigating the chemical coding of nerve structures in the wall of the ileum and large intestine in normal pigs (n = 3) and in pigs undergoing dysentery (n = 6). Dysentery was evoked by artificial infection of the clinically healthy animals per os with Brachyspira hyodysenteriae. All the animals were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde. The cryostat sections of the intestines were processed for double‐labelling immunohistochemistry using antisera against PGP 9.5, GAL and VIP. In the intramural plexuses of the control pigs, the percentage of GAL‐immunoreactive (GAL‐IR) perykarya varied from 11% (descending colon) to 19% (centrifugal turns of the ascending colon) whereas in the dysenteric pigs, it was distinctly higher, reaching from 28% (ileum) up to 48% (cecum). In the control animals, the percentage of VIP‐IR neuronal somata varied from 3% (descending colon) to 19% (ileum). In dysenteric pigs, it was from 6% (descending colon) up to 28% (cecum). In the muscular coat (MC) and mucous membrane (MM) of the normal intestine, very numerous GAL‐ and VIP‐IR nerve fibres were observed. The nerve fibres in the myenteric plexus (MP) were even more numerous than those in the muscular coat while in the outer (OSP) and inner (ISP) submucous plexuses, they were less abundant. In the dysenteric pigs, the nerve fibres found in MC, MP and OSP were less numerous, whereas those observed in ISP and MM were more abundant than those in the control animals. The present results suggest that GAL and VIP are involved in the regulation of inflammatory processes developing in the porcine gastrointestinal tract during dysentery.     

Fractures of Ossified Rib Cartilages in the Horse – a Concern of Veterinarians?

The morphology, neurochemistry and function of intramural nerve structures in the mammalian gastrointestinal tract are relatively well known, but in normal, healthy individuals. The present study was aimed at investigating the chemical coding of nerve structures in the wall of the ileum and large intestine in normal pigs ( n  = 3) and in pigs undergoing dysentery ( n  = 6). Dysentery was evoked by artificial infection of the clinically healthy animals per os with Brachyspira hyodysenteriae . All the animals were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde. The cryostat sections of the intestines were processed for double-labelling immunohistochemistry using antisera against PGP 9.5, GAL and VIP. In the intramural plexuses of the control pigs, the percentage of GAL-immunoreactive (GAL-IR) perykarya varied from 11% (descending colon) to 19% (centrifugal turns of the ascending colon) whereas in the dysenteric pigs, it was distinctly higher, reaching from 28% (ileum) up to 48% (cecum). In the control animals, the percentage of VIP-IR neuronal somata varied from 3% (descending colon) to 19% (ileum). In dysenteric pigs, it was from 6% (descending colon) up to 28% (cecum). In the muscular coat (MC) and mucous membrane (MM) of the normal intestine, very numerous GAL- and VIP-IR nerve fibres were observed. The nerve fibres in the myenteric plexus (MP) were even more numerous than those in the muscular coat while in the outer (OSP) and inner (ISP) submucous plexuses, they were less abundant. In the dysenteric pigs, the nerve fibres found in MC, MP and OSP were less numerous, whereas those observed in ISP and MM were more abundant than those in the control animals. The present results suggest that GAL and VIP are involved in the regulation of inflammatory processes developing in the porcine gastrointestinal tract during dysentery.     

Immunohistochemical study of the otic ganglion in the pig

The presence of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in neurons and nerve fibers of the porcine otic ganglion. ChAT-positive neurons were very numerous while VAChT-positive nerve cells were moderate in number. The number of neurons containing NPY and VIP was lower and those containing SOM, GAL, SP or CGRP were observed as scarce, or single nerve cells. The above mentioned substances (except SOM) were present in nerve fibers of the ganglion. ChAT- and VAChT-positive nerve fibers were numerous, while the number of nerve terminals containing NPY, VIP and SP was lower. GAL- and CGRP-positive nerve fibers were scarce.     

Immunohistochemical characterization of neurons in the porcine ciliary ganglion

The present study was aimed at disclosing the chemical coding of nerve structures in the porcine ciliary ganglion (CG) using immunohistochemical methods. The substances under investigation included markers of "classical" neurotransmitters, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH) as well as neuropeptides, somatostatin (SOM), galanin (GAL), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Immunoreactivity to ChAT and VAChT was found virtually in all the neuronal somata and in numerous intraganglionic, varicose nerve fibres which often formed basket-like formations around the nerve cell bodies. Many CG neurons contained immunoreactivity for SOM (46%) or GAL (29%). Interestingly, a small number (approx. 1%) of the cholinergic somata stained for TH but not for DbetaH; nevertheless, some extra- and intraganglionic nerve fibres displayed immunoreactivity for DbetaH or TH. The CG perikarya stained neither for vasoactive intestinal polypeptide (VIP) nor for neuropeptide Y (NPY), but some NPY- or VIP-positive nerve terminals were observed within nerve bundles distributed outside the ganglion. SP- and CGRP-immunoreactivity was found in some intraganglionic nerve fibres only. The present study revealed that the porcine CG consists of cholinergic neurons many of which contain SOM and GAL. Thus, it can be assumed that in the pig, these neuropeptides are involved, complementary to acetylocholine, in the parasympathetic postganglionic nerve pathway to structures of the eye including the ciliary and iris sphincter muscles.     

Analysis of the chemical coding of neurons in the intermediate thoracic ganglion of the pig

The pig has been widely used as a model in cardiovascular research. A unique feature of the porcine extrinsic sympathetic cardiac nerves is that they arise from intermediate ganglia in the thoracic cavity. The localization and pattern of distribution of nerve cell bodies and fibers containing tyrosine hydroxylase (TH), dopamine B-hydroxylase (DBH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET) as well as calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was studied with immunohistochemistry. Almost all the neurons showed immunoreactivity to TH. Immunoreactivity to NPY, VIP, SOM, GAL, MET and PACAP was displayed by nerve cell bodies while nerve fibers exhibited immunoreactivity to all the neuropeptides studied. Therefore, it seems that the chemical coding of neurons and especially nerve fibers in the porcine intermediate ganglion share general similarities (with certain neurochemical variability), with porcine prevertebral ganglia (e.g., celiacomesenteric and caudal mesenteric ganglia).     

不同日龄仔猪小肠黏膜下神经丛神经元类型变化的研究

采用组织化学和免疫组织化学的方法对0、5、28日龄的仔猪小肠黏膜下神经丛中神经元类型的变化进行了定性研究和定量测定。结果表明，仔猪小肠黏膜下神经丛分为靠近环肌层表面的ESP(external submucosal plexus)和靠近黏膜肌层的ISP(internal submucosal plexus)；ESP与ISP神经纤维的性质不同，ESP主要是有髓神经纤维，而ISP主要是无髓神经纤维；随着日龄增长，黏膜下神经丛发育较快，ESP与ISP的差异日益显著。研究揭示：仔猪出生后黏膜下神经丛在形态和不同亚群神经元比例上均有一调整期，其生理学意义可能是使小肠黏膜下神经丛的功能日益完善。     

An immunohistochemical study of various peptide-containing endocrine cells and neurones at the equine ileocaecal junction

The ileocaecal junctions of 5 horses and 2 donkeys were examined by using antisera to the following peptides: somatostatin, glucagon, gastrin, neurotensin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY). Antisera to somatostatin, neurotensin and NPY demonstrated endocrine cells in the ileal- and caecal parts of the ileocaecal junction, while immunoreactivity for glucagon was demonstrated in endocrine cells of the ileal part only. Nerve cell bodies showing immunoreactivity to SP, VIP, CGRP and PHI were demonstrated in the myenteric and submucosal plexuses and were associated with small blood vessels in the submucosa of all the regions tested. Ramified nerve fibres in the submucosa immunoreactive to SP, VIP, CGRP and PHI extended to the mucosa and to small blood vessels in the submucosa. Nerve fibres showing immunoreactivity to SP, VIP and PHI extended to the circular smooth muscle layer of the ileocaecal junction.     

Calcium-Binding Proteins and Neurotransmitters in the Stomach Myenteric Plexus of the Korean Native Goat

The expression of calbindin D-28k (CB), calretinin (CR), substance P (SP) and calcitonin gene-related peptide (CGRP) in the stomach myenteric plexus of the Korean native goat stomach was investigated by immunohistochemistry. The results demonstrated the presence of nerve fibers and cell bodies immunoreactive (IR) to CB, CR, SP and CGRP. In tissues of rumen, reticulum, omasum and abomasum, some distinct neuronal populations could be distinguished according to their morphologic and neuronal chemical properties: Dogiel type I cells which have irregular lamellar dendrites and a single axon, Dogiel type II cells which have large ovoid cell bodies and several long axon-like processes, and small filamentous interneurons. CB-, CR-, SP- and CGRP-IR neurons and fibers were observed in the myenteric plexus of stomach, and varicose nerve fiber immunostained to SP and CGRP also were found in the muscle layer. In myenteric plexus of the stomach, CB- and SP-positive neurons were characterized by Dogiel type II and CR-IR neurons were classified Dogiel type I with lamellar dendrites, and immunoreactivity of CGRP was very weak in the somata. SP- and CGRP-IR nerve fibers formed dense networks within the myenteric ganglia. SP-IR cell bodies and their fibers were found in the myenteric plexus, and the immunoreactivity and number of cell bodies were more than CB-, CR-, and CGRP-IR neurons. These results suggest that SP, CGRP, CB and CR in the myenteric neurons of Korean native goat stomach may have play an important role in the dynamic movement.
(Support contributed by: Korean Research Foundation 2003-015-E00195).     

Immunohistochemical characteristics of neurons supplying the porcine bulbourethral gland

In the male pig, the bulbourethral gland (BG) is a particulary well developed accessory genital gland (AGG) which produces complex secretion contributing to the fluid component of semen. The secretory and motor function of AGGs is thought to be under the autonomic nervous system control. Although relatively much is known about the innervation of the prostate gland and, to a lesser degree, of the seminal vesicle, the paucity of data dealing with the innervation of BG is striking. Therefore, combined retrograde tracing and double-labelling immunofluorescence have been used to investigate the distribution and immunohistochemical properties of autonomic and primary afferent neurons projecting to this gland in the pig. BG-projecting neurons were found in some ipsilateral (I) and contralateral (C) sympathetic chain ganglia (SChG), the caudal mesenteric ganglion (CaMG), pelvic ganglia (PG) and some dorsal root ganglia (DRG). Immunohistochemistry revealed that the vast majority of CaMG and SChG BG-projecting neurons contained tyrosine hydroxylase (TH) and dopaminebeta-hydroxylase (DbetaH), and some neuropeptides including neuropeptide Y (NPY), somatostatin (SOM) and galanin (GAL). Three subpopulations of PG neurons supplying BG could be distinguished: 1) cholinergic neurons [vesicular acetylcholine transporter (VAChT)-positive] which also contained vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), SOM and NPY, 2) adrenergic neurons (TH-positive) which also stained for NPY, GAL or leu5-enkephalin (LEU), and 3) non-adrenergic, non-cholinergic neurons (NANC). DRG BG-projecting neurons contained mostly substance P (SP) and/or calcitonin gene-related peptide (CGRP) which sometimes colocalized with GAL. The possible functional significance of the substances found within the neurons is discussed.     

Immunohistochemical localization of calcium binding proteins and some neurotransmitters in myenteric plexus of goat stomach

To understand the neurochemical properties of the gastric myenteric plexus of ruminants, the expression patterns of calbindin D-28k (CB), calretinin (CR), substance P (SP) and calcitonin gene-related peptide (CGRP) were explored in the Korean native goat. In gastric myenteric plexus, CB and SP immunoreactivity were observed in round- or oval-shaped neurons. CR and CGRP immunoreactivity were detected only in the nerve fibers. This immunohistochemical localization of CB, CR, CGRP and SP in the myenteric plexus of the goat stomach exhibited species-specific patterns. These findings suggest that these substances may be directly or indirectly related to the gastric functions of the goat stomach.     

Effect of total or partial uterus extirpation on sympathetic uterus-projecting neurons in porcine inferior mesenteric ganglion. B. Changes in expression of neuropeptide Y,galanin, vasoactive intestinal polypeptide,pituitary adenylate-cyclase activating peptide,somatostatin and substance P

The expression of neuropeptide Y (NPY), galanin (GAL), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM) and substance P (SP) was studied in the neurons of the inferior mesenteric ganglion (IMG) projecting to the uterine horn and uterine cervix after uterus extirpation-induced axotomy in sexually immature gilts. The expression was studied with immunohistochemistry, in situ hybridization and RT-PCR. Uterus-projecting neurons were identified by retrograde tracing with Fast Blue (FB). Immunohistochemistry revealed that FB-positive (FB+) uterus-projecting neurons in control animals contained only immunoreactivities to NPY (ca. 50%) and GAL (single neurons). Uterus extirpation increased the occurrence of NPY and GAL in FB+ neurons. No other studied neuropeptides were found in axotomized uterus-projecting neurons. Hybridization in situ revealed the reduction of NPY expression and induction of GAL expression in FB+ neurons. RT-PCR detected induction of GAL expression in the IMG after uterus extirpation. The expression level of NPY and SOM was significant and was not affected by axotomy. The expression level of PACAP was very low and did not differ between IMG of control, partially and totally hysterectomized animals. No VIP and SP expression was detected in all ganglia. The presented data show clear axotomy-related changes in the expression of GAL and NPY in the uterus-projecting neurons of the porcine IMG.     

Histochemical study of the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons in the chicken caecum

The distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurones was investigated in the chicken caecum. Double staining combined NADPHd histochemistry with immunohistochemistry for neural nitric oxide synthase (nNOS) indicated that NADPHd-positive neurones also showed immunoreactivity for nNOS. NADPHd-positive nerve cell bodies were observed in both the myenteric and the submucous plexuses. Nerve fibres showing enzyme activity were mainly distributed in the circular muscle layer, but only a few fibres in the mucosal layer. Fine nerve fibres showing NADPHd activity were found running between germinal centres in the caecal tonsil. Quantitative analysis showed no significant differences in the number of enzyme-positive nerve cell bodies per ganglion of the myenteric and the submucous plexuses among three different caecal regions; proximal, middle and distal regions. Larger numbers of ganglia were detected in the submucous plexus than the myenteric plexus at all three regions. These data indicate that nitrergic neurones in the submucous plexus mainly project to the circular muscle layer in the chicken caecum. It is possible that nitrergic nerves regulate the motility of the chicken caecum.     

Immunohistochemical study of the pre- and postnatal innervation of the dog lower urinary tract: morphological aspects at the basis of the consolidation of the micturition reflex

Immunohistochemical studies were performed on male and female bladder and urethra collected from 4 adults dogs and 10 foetal specimens with crown-rump length from 53 to 155 mm (medium-sized breeds, presumptive 38 days of gestation to term). A panel of antisera was tested, including PGP 9.5 to describe the general intramural innervation, ChAT and TH to depict the cholinergic and nor-adrenergic components and NOS1, CGRP, SP, NPY, VIP, SOM, GAL, 5-HT to investigate the possible nitrergic, peptidergic and aminergic ones. A rich cholinergic innervation was present in adult bladder and urethra, along with a lesser number of adrenergic nerves and a small number of nitrergic ones. Either bladder or urethra received numerous CGRP-, SP-, NPY-, VIP-containing nerve fibres which were distributed throughout the muscle layers. All over the lower urinary tract strong to weak ChAT-, CGRP-, SP- and NPY-immunoreactivity was detected in intramural ganglia, in peripheral nerve bundles and around blood vessels. 5-HT-immunoreactive endocrine cells were present in the urethral epithelium. Early foetal organs were supplied only by cholinergic nerve fibres. Few NOS-, CGRP- and SP-ergic components appeared at the end of pregnancy. It can be guessed that sensory mediators such as CGRP and SP increase in postnatal ages while other neuropeptides, such as NPY and VIP, appear only after birth, as the urinary reflex consolidates.     

Lesions of the enteric nervous system and the possible role of mast cells in the pathogenic mechanisms of migration of schistosome eggs in the small intestine of cattle during Schistosoma bovis infection

The enteric nervous system in the small intestine of cattle during Schistosoma bovis infection was studied by histological stains and immunohistochemical methods. Lesions due to migration of schistosoma eggs were located mainly in the mucous and the submucous layer overlaying the submucous vascular arcades. Granulomas destroyed ganglia, neurons, nerves fibre strands and nerve fibres. Ganglia situated within or near granulomas were infiltrated by mast cells, eosinophils, lymphocytes, globule leukocytes, neutrophils and macrophages. Mast cells were in close contact with degenerating neuronal perikarya. Whereas vasoactive intestinal peptide-like immunoreactivity in the nerves and neurons in the ganglia within and around granulomas was increased, the neurofilament-like immunoreactivity was reduced. Compared to the myenteric and external submucous plexuses, the internal submucous and mucous plexuses were the most damaged. These changes imply reduced functional capacity in the nervous tissue which might cause reduced motility, malabsorption and partly account for the loss of body weight and condition and failure to thrive which occur in schistosomosis.Biotinylated affinity purified swine anti-rabbit and mouse anti-rabbit immunoglobulins reacted nonspecifically with a subset of mast cells. The reaction revealed many mast cells in early forming granulomas and around schistosome egg tracts and infiltration of mast cells into the ganglia of intestinal nerve plexuses. The observation shows a localized, Type I hypersensitivity reaction suggesting for the release of mast cell-derived chemical mediators in the intestinal reaction to trap or evict S. bovis eggs and to cause diarrhoea.