Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon—a quantitative immunohistochemical study

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas’ disease.     

Chagasic megacolon: enteric neurons and related structures

Megacolon, the irreversible dilation of a colonic segment, is a structural sign associated with various gastrointestinal disorders. In its hereditary, secondary form (e.g. in Hirschsprung’s disease), dilation occurs in an originally healthy colonic segment due to an anally located, aganglionic zone. In contrast, in chronic Chagas’ disease, the dilated segment itself displays pathohistological changes, and the earliest and most prominent being found was massive loss of myenteric neurons. This neuron loss was partial and selective, i.e. some neurons containing neuronal nitric oxide synthase and/or vasoactive intestinal peptide (VIP) were spared from neuron death. This disproportionate survival of inhibitory neurons, however, did not completely correlate with the calibre change along the surgically removed, megacolonic segments. A better correlation was observed as to potentially contractile muscle tissue elements and the interstitial cells of Cajal. Therefore, the decreased densities of α-smooth muscle actin- and c-kit-immunoreactive profiles were estimated along resected megacolonic segments. Their lowest values were observed in the megacolonic zones itself, whereas less pronounced decreases were found in the non-dilated, transitional zones (oral and anal to dilation). In contrast to the myenteric plexus, the submucosal plexus displayed only a moderate neuron loss. Neurons co-immunoreactive for VIP and calretinin survived disproportionately. As a consequence, these neurons may have contributed to maintain the epithelial barrier and allowed the chagasic patients to survive for decades, despite their severe disturbance of colonic motility. Due to its neuroprotective and neuroeffectory functions, VIP may play a key role in the development and duration of chagasic megacolon.     

What neurons hide behind calretinin immunoreactivity in the human gut?

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.     

Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons

The submucous layers of human small and large intestines contain at least two separate neuron populations. Besides morphological features, they differ in their immunoreactivities for calretinin (CALR) and somatostatin (SOM), respectively. In this study, submucosal wholemounts of 23 patients or body donors (including all segments of small intestine and colon) were immunohistochemically quadruple stained for CALR and SOM as well as for substance P (SP) and choline acetyltransferase (ChAT). We found that all SOM-positive neurons co-stained for ChAT and the majority for SP [between 50 % in the small intestinal external submucosal plexus (ESP) and 75 % in the colonic ESP]. In contrast, a majority of CALR-neurons contained ChAT (between 77 % in the small intestinal ESP and 92 % in the large intestinal ESP) whereas less than 4 % of CALR-neurons were co-immunoreactive for SP. Another set of wholemounts was co-stained for peripherin, a marker enabling morphological analysis. Where identifiable, both SOM alone- and SOM/SP-neurons displayed a uniaxonal (supposed pseudouniaxonal) morphology. We suggest that the chemical code of SOM-immunoreactive, human submucosal neurons may be “ChAT+/SOM+/SP±”. In additional sections double stained for SOM and SP, we regularly found double-labelled nerve fibres only in the mucosa. In contrast, around submucosal arteries mostly SOM alone- fibres were found and the muscularis propria contained numerous SP-alone fibres. We conclude that the main target of submucosal SOM(/SP)-neurons may be the mucosa. Due to their morpho-chemical similarity to human myenteric type II neurons, we further suggest that one function of human submucosal SOM-neurons may be a primary afferent one.     

Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine

This study aimed at estimating the proportion of human myenteric Dogiel type II neurons, putative intrinsic primary afferent neurons (IPANs), in relation to the entire myenteric neuron population. Since, at present, there is no known single marker, which specifically labels these neurons, we tried to identify the most appropriate marker combination based on the results of an earlier study. For this purpose, 10 wholemounts derived from human small intestinal segments were immunohistochemically triple-stained for calretinin (CALR), somatostatin (SOM) and neurofilaments (NF) and 9 were stained for substance P (SP), SOM and NF. In each wholemount, 15 ganglia selected randomly were evaluated. On the basis of their NF-reactivity, neurons reactive for one or co-reative for both of the other two markers, respectively, were morphologically classified as type II or non-type II neurons. We found that the majorities of neurons co-reactive for CALR/SOM and SP/SOM, respectively, were type II neurons whereas this was not the case for neurons, which were reactive for only one of the two markers. One of the statistical parameters estimated was the positive predictive value, the probability that a neuron displaying CALR/SOM- or SP/SOM-co-reactivity, respectively, is a type II neuron. This value was 97% in case of CALR/SOM- and 95% in case of SP/SOM-co-staining. Although the difference of the statistical parameters between the two stainings was not significant, CALR and SOM were used to estimate indirectly the proportion of type II neurons, in wholemounts co-stained with the pan-neuronal marker neuronal protein HuC/HuD (HU). In these wholemounts, altogether 9.1% of neurons were coreactive for CALR/SOM. We suggest that the proportion of myenteric type II neurons in the human small intestine is related to the proportion of CALR/SOM-co-reactive neurons and may be near to one tenth of the total myenteric neuronal population.     

Partial,selective survival of nitrergic neurons in chagasic megacolon

One frequent chronic syndrome of Chagas’ disease is megacolon, an irreversible dilation of a colonic segment. Extensive enteric neuron loss in the affected segment is regarded as key factor for deficient motility. Here, we assessed the quantitative balance between cholinergic and nitrergic neurons representing the main limbs of excitatory and inhibitory colonic motor innervation, respectively. From surgically removed megacolonic segments of four patients, each three myenteric wholemounts (from non-dilated oral, megacolonic and non-dilated anal parts) was immunohistochemically triple-stained for choline acetyltransferase, neuronal nitric oxide synthase (NOS) and the panneuronal human neuronal protein Hu C/D. Degenerative changes were most pronounced in the megacolonic and anal regions, e.g. bulked, honeycomb-like ganglia with few neurons which were partly enlarged or atrophic or vacuolated. Neuron counts from each 15 ganglia of 12 megacolonic wholemounts were compared with those of 12 age- and region-matched controls. Extensive neuron loss, mainly in megacolonic and anal wholemounts, was obvious. In all three regions derived from megacolonic samples, the proportion of NOS-positive neurons (control: 55%) was significantly increased: in non-dilated oral parts to 61% (p = 0.003), in megacolonic regions to 72% (p < 0.001) and in non-dilated anal regions to 78% (p < 0.001). We suggest the chronic dilation of megacolonic specimens to be due to the preponderance of the nitrergic, inhibitory input to the intestinal muscle. However, the observed neuronal imbalance was not restricted to the dilated regions: the non-dilated anal parts may be innervated by ascending, cholinergic axons emerging from less affected, more anally located regions.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

Nitric oxide synthase in the gastrointestinal tract of the estuarine crocodile, Crocodylus porosus

The aim of this study was to investigate the distribution of nitric oxide synthase (NOS)-containing nerve cells in the gastrointestinal tract of a reptile and to compare it with the pattern in other vertebrate classes. In the estuarine crocodile, Crocodylus porosus, NOS-positive nerve cell bodies and fibres were found in all regions of the gut examined. Most myenteric microganglia contained one or several NOS-immunoreactive neurons together with unlabelled neurons. The majority of the neurons were multipolar, ranging from 10 to 25 microns in diameter. Both the circular and the longitudinal muscle layers were innervated by NOS-immunoreactive nerve fibres, which mostly ran parallel to the muscle fibres. In addition, small blood vessels in the submucosa and on the serosal surface of the gut were innervated by NOS-immunoreactive fibres. Double labelling with antisera to NOS and vasoactive intestinal peptide (VIP) revealed three neuronal subpopulations. A small proportion of the NOS-immunoreactive cells also contained immunoreactivity to VIP while a majority of the VIP-immunoreactive cells were NOS immunoreactive. There were more nerve fibres showing VIP immunoreactivity than fibres with NOS immunoreactivity, although most of the latter also contained immunoreactivity to VIP. VIP-immunoreactive fibres often surrounded the NOS-immunoreactive nerve cells. These results suggest that neuronally released nitric oxide is likely to be involved in the control of gastrointestinal motility in the crocodile as in most other vertebrate species.     

Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig

Summary The distributions within the coeliac ganglion of different chemically coded subgroups of noradrenaline neurons, and the relationships between these neurons and nerve fibres projecting to the ganglion from the intestine, have been assessed quantitatively by use of an immunohistochemical double-staining method. Noradrenaline (NA) neurons made up 99% of all cell bodies. Of these, 21% were also reactive for somatostatin (NA/SOM neurons), 53% were also reactive for NPY (NA/NPY neurons), and 26% were not reactive for either peptide. NA neurons without reactivity for any of the peptides whose localization was tested have been designated NA/-. A small percentage, about 1%, of neurons were reactive for both NPY and SOM. The three major types of NA neurons were arranged in clumps or ribbons throughout the ganglia, with a tendency for NA/SOM neurons to be medial and NA/NPY neurons to be lateral in the ganglia. A small group of neurons (<1%) encoded with dynorphin, NPY and vasoactive intestinal peptide (VIP) was encountered. VIP-immunoreactive nerve terminals, projecting to the ganglion from cell bodies in the intestine, ended around NA/SOM and NA/neurons but not around NA/NPY neurons. Thus, the VIP axons from the intestine end selectively around neurons that modify intestinal function (NA/SOM and NA/-neurons) but not around neurons, the terminals of which supply blood vessels (NA/NPY neurons).     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Trypanosoma cruzi: preliminary investigation of NADH-positive and somatostatin-immunoreactive neurons in the myenteric plexus of the mouse colon during the infection

In this paper, the distribution of NADH-positive and somatostatin (SOM) immunoreactive neurons in the myenteric plexus of the colon of mice infected with Trypanosoma cruzi was studied. Ten young, male, BALB/c mice were inoculated with the Y strain of T. cruzi, 60 days previously (chronic phase of the infection). Another 10 mice were uninfected controls. Distal and proximal colonic neurons from five chronically infected mice and their controls were stained using the NADH-diaphorase method. Quantitative results showed a significant decrease of 39% in the number of neurons in the proximal colon of infected mice and 58% in the distal colon (p<0.05). SOM was localized in five animals from each group by light microscopy, using an indirect immunofluorescence technique. It was observed that there were far fewer nerve cells and fibres and less intensely stained neuron bodies and varicose SOM-positive nerve fibres in both, control and chronic infected mice. These findings could be related to the disturbances in intestinal motility observed in patients in the chronic phase of Chagas' disease.     

Vascular control of the pig nasal mucosa: distribution and effect of somatostatin in relation to noradrenaline and neuropeptide Y

By means of immunohistochemistry and radioimmunoassay (RIA), we have investigated the possible occurrence of somatostatin (SOM)-like immunoreactivity (-LI) in the autonomic innervation of the pig nasal mucosa. SOM-immunoreactive (-IR) fibres were present around nasal arteries, arterioles and venous sinusoids. Double-labelling experiments revealed that SOM-LI was co-localized with the noradrenaline (NA) markers tyrosine hydroxylase and dopamine-β-hydroxylase as well as with neuropeptide Y (NPY) in a subpopulation of neurons in the superior cervical sympathetic ganglion and in perivascular nerve terminals. Furthermore, SOM-LI was also present in perivascular fibres containing vasoactive intestinal polypeptide (VIP) and NPY of presumably parasympathetic origin. The parasympathetic fibres that were associated with glands contained peptide histidine isoleucine (PHI), VIP and NPY but not SOM, suggesting that in the nasal mucosa SOM-IR is restricted to perivascular nerves. As revealed by RIA, the content of SOM-LI in biopsies of both nasal mucosa and superior cervical sympathetic ganglion was about 12 pmol/g and the reverse phase HPLC characterisation of SOM-LI shown two separate peaks for SOM-28 and SOM-14.     

Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways

Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Two submucosal nerve plexus in human intestines

We examined the architecture of human submucosal nerve networks of gut segments derived from 12 individuals (each six from small and large intestines). Twelve undivided submucosal wholemounts were prepared and immunohistochemically stained for peripherin (nerve elements) and for α-smooth muscle actin (remnants of attached muscle bundles). We found two ganglionic nerve networks. The plexus submucosus externus was generally monolayered and located under the outermost surface of the submucosal wholemounts. Its nerve fibre strands frequently joined each other in acute or obtuse angles, the meshes of the network were relatively wide and frequently polyangular shaped. The plexus submucosus internus was generally multi-(mostly two- or three-)layered and occupied at least the inner half of the thickness of the wholemount, sometimes extending abluminally beyond the great submucosal vessels. Its meshes were irregular. The shapes of ganglia of the two plexus were generally different, those of the internal plexus were frequently grape-like whereas the neurons of external ganglia were mostly embedded in the contoures of the joining nerve fibres. Both plexus were intensely connected via coiled interconnecting strands, either with or without intercalated ganglia. For use of eponyms for two different submucosal plexus, the names of Meissner (inner) and Schabadasch (outer) are historically justified.     

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.