J.-P. Timmermans

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract.

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

TRPV1 receptors on unmyelinated C‐fibres mediate colitis‐induced sensitization of pelvic afferent nerve fibres in rats

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis‐induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single‐fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N‐(4‐tertiarybutylphenyl)‐4‐(3‐chlorophyridin‐2‐yl)tetrahydropyrazine‐1(2H)carboxamide (BCTC; 0.25–5 mg kg−1) or its vehicle (hydroxypropyl‐β‐cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A‐fibre neurons, was performed in the dorsal root ganglia L6–S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C‐fibres. BCTC did not significantly affect the C‐fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C‐fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Aδ‐fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up‐regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis‐induced sensitization of pelvic afferent C‐fibres to CRD, while Aδ‐fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Role of oxidative stress in the pathogenesis of septic ileus in mice.

Abstract  We investigated the role of oxidative stress in the pathogenesis of septic ileus. Sepsis was induced by intraperitoneal (i.p.) injection of lipopolysaccharides (LPS, 20 mg kg−1) in mice. The effect of two i.p. injections of superoxide dismutase [polyethylene glycol (PEG)‐SOD, 4000 U kg−1] and catalase (PEG‐CAT, 15 000 U kg−1) was investigated on gastric emptying, intestinal transit and total nitrite plasma concentrations. We also performed immunohistochemical experiments on gastric and ileal tissue. LPS significantly delayed gastric emptying and intestinal transit while plasma nitrite levels increased. Polyethylene glycol (PEG)‐SOD reversed the endotoxin‐induced delay in gastric emptying and improved the delay in intestinal transit without effect on plasma nitrite levels. PEG‐CAT slightly improved the delay in gastric emptying without effect on intestinal transit. Immunohistochemistry showed the presence of nitrotyrosine (NT) and 4‐hydroxy‐2‐nonenal (HNE) in the gastric and ileal mucosa of LPS‐treated mice. Treatment with PEG‐SOD or PEG‐CAT of LPS mice diminished the presence of NT or HNE in both tissues. In addition, LPS induced a significant increase in inducible nitric oxide synthase (iNOS)‐positive residential macrophages in the external musculature of stomach and ileum, which significantly decreased after PEG‐SOD or PEG‐CAT treatment. The present results support a role for oxidative and nitrosative stress in the pathogenesis of septic ileus in mice.

Selective inhibition of FAAH produces antidiarrheal and antinociceptive effect mediated by endocannabinoids and cannabinoid-like fatty acid amides

The endogenous cannabinoid system (ECS) plays a crucial role in multiple physiological processes in the central nervous system and in the periphery. The discovery that selective cannabinoid (CB) receptor agonists exert a potent inhibitory action on gastrointestinal (GI) motility and pain has placed the ECS in the center of attention as a possible target for the treatment of functional GI diseases. However, side effects of CB agonists prompted the search for novel therapeutic targets. Here, the effect of PF‐3845, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor in the GI tract was investigated.

Calcitonin Gene-Related Peptide-Like Immunoreactivity in the Human Small Intestine

Calcitonin-gene-related-peptide (CGRP)-like immunoreactivity was localized in nerve fibres, neuronal somata and in mucosal endocrine cells of the human small intestine. Immunoreactive enteric neurons were more numerous in the submucous plexuses than in the myenteric plexus. Morphologically, they predominantly had the appearance of type II neurons. The majority of the CGRP-like immunoreactive nerve fibres ran within the ganglionic nerve plexuses. Only a small proportion could be observed in the lamina propria, the lamina muscularis mucosae, or the circular and longitudinal outer smooth muscle layer. These findings suggest that within the wall of the human small intestine neuronal CGRP of either extrinsic or intrinsic origin exerts its effect chiefly on other enteric neurons, and might be indirectly involved in the regulatory functions of the human small intestine.

Occurrence, distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.

NO-Synthase-Containing Neurons of the Pig Inferior Mesenteric Ganglion, Part of Them Innervating the Ductus deferens

The presence of nitric oxide-synthase (NOS) in neurons of the porcine inferior mesenteric ganglion (IMG) has been investigated. A minority (about 1-3%) of the neurons were immunoreactive (IR) for NOS, the vast majority of which stained for neuropeptide Y (NPY) but not for tyrosine hydroxylase (TH). A small subpopulation of prevertebral neurons, 1% of which stained for NOS or NADPH-diaphorase (NADPHd), projected to the ductus deferens, as demonstrated by retrograde tracing. Within the wall of the ductus deferens, NOS- or NOS/NPY-IR nerve fibres were found to innervate the smooth muscle or were closely associated with blood vessels. It is therefore suggested that nitric oxide might be involved in the regulation of local blood flow and muscular tone in the wall of the pig ductus deferens.

Immunohistochemical localization of calbindin-D28K in the brain of a cartilaginous fish, the dogfish (Scyliorhinus canicula L.).

The occurrence and distribution of the vitamin-D-induced calcium-binding protein, calbindin-D28K, has been studied in the brain of a cartilaginous fish using immunohistochemical techniques. A strong immunoreactivity was found in the perikarya, dendrites and axons of neurons located in the nucleus interstitialis commissurae anterioris, the nucleus medialis of the left habenula, the thalamus dorsalis, the thalamus ventralis, the nucleus lobi lateralis, the nucleus interpeduncularis, the lobus vagi and the medial reticular zone. Fibre tracts associated with some of these neuronal groups, such as the fasciculus retroflexus, the stria medullaris and the commissura habenulae, also contained immunopositive fibres. Only a minor immunoreactivity could be detected in other brain areas such as the tectum mesencephali and some telencephalic zones. Interestingly, the cerebellum did not show any immunoreactivity in Purkinje cells nor in other neurons. The distribution of calbindin-D28K in the dogfish brain appears to be mainly related to the viscerosensory centres.

Effect of intestinal inflammation on the cell-specific expression of somatostatin receptor subtypes in the murine ileum.

Abstract  Despite our knowledge of somatostatin (SOM) in gastrointestinal functions, little information is available on the SOM receptors (SSTRs) mediating these effects. This study focussed on the expression of SSTRs in non‐inflamed and Schistosoma mansoni‐infected murine ileum using immunocytochemistry, reverse transcriptase (RT)‐PCR and quantitative real time RT‐PCR (qPCR). In the non‐inflamed ileum, SSTRs showed a widespread, cell‐type specific expression pattern. For instance, SSTR2A immunoreactivity was detected in a minor population of submucous but not myenteric glial cells. In the inflamed ileum, significant changes in the expression pattern of SSTRs occurred, with SSTR1 and SSTR3 expression on mucosal mast cells (MMCs) and mucosal nerve fibres. SSTR4‐immunoreactive nerve fibres were detected in granulomas and the lamina propria. qPCR experiments indicated significantly increased mRNA levels for SOM, SSTR1 and SSTR3 in inflamed ileum. This study reveals that SSTRs are expressed in specific cell types in murine ileum. Expression of SSTR1 and SSTR3 on MMCs and increased density of SOM‐expressing nerve fibres in the lamina propria during inflammation, support the hypothesis that SOM is implicated in the physiological control of MMCs during intestinal inflammation. Evidence is provided that in mouse mainly SSTR1, SSTR3 and SSTR4 are involved in the somatostatinergic inflammatory effects during intestinal schistosomiasis.

Role of reactive nitrogen species in neuronal cell damage during intestinal schistosomiasis

Abstract. Free radicals are known to be involved in the host reaction during Schistosoma mansoni-induced inflammation in the liver and the intestine. In the present study, the influence of reactive nitrogen species (RNS) on the enteric neurons of infected ileum of mice was investigated. Cryosections and whole-mounts of the ileum of control, and 8- and 15-week-infected mice were processed for immunohistochemical localization of 3-nitrotyrosine, a biomarker of RNS, and of active caspase-3, a key executioner of apoptosis. An antibody directed against protein gene product 9.5 or S100 protein was used as a marker for neurons or enteroglial cells. In infected mice, but not in control animals, 3-nitrotyrosine was detected in parasite eggs and, as revealed by double immunolabelling, in some neuronal and enteroglial cells. Quantitative analysis of whole-mounts showed that the percentage of 3-nitrotyrosine-immunoreactive neurons significantly increased with time in both the submucous and myenteric plexus. Caspase-3 immunoreactivity was predominantly found in parasite eggs in infected mice. Immunoreactive enteric neurons were occasionally observed. The results indicate that inflammation-induced RNS are present in the ileum of S. mansoni-infected mice, and participate in the elimination of the schistosome eggs causing damage in a significant number of enteric neurons. However, neuronal cell death appears to be a rare phenomenon in the schistosome-infected mouse ileum.