Evidence for long-term sensitization of the bowel in patients with post-infectious-IBS.

Post-infectious irritable bowel syndrome (PI-IBS) is a common gastrointestinal disorder characterized by persistent abdominal pain despite recovery from acute gastroenteritis. The underlying mechanisms are unclear, although long-term changes in neuronal function, and low grade inflammation of the bowel have been hypothesized. We investigated the presence and mechanism of neuronal sensitization in a unique cohort of individuals who developed PI-IBS following exposure to contaminated drinking water 7 years ago. We provide direct evidence of ongoing sensitization of neuronal signaling in the bowel of patients with PI-IBS. These changes occur in the absence of any detectable tissue inflammation, and instead appear to be driven by pro-nociceptive changes in the gut micro-environment. This is evidenced by the activation of murine colonic afferents, and sensitization responses to capsaicin in dorsal root ganglia (DRGs) following application of supernatants generated from tissue biopsy of patients with PI-IBS. We demonstrate that neuronal signaling within the bowel of PI-IBS patients is sensitized 2 years after the initial infection has resolved. This sensitization appears to be mediated by a persistent pro-nociceptive change in the gut micro-environment, that has the capacity to stimulate visceral afferents and facilitate neuronal TRPV1 signaling.

NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.

A qualitative exploration of the collaborative working between palliative care and geriatric medicine: Barriers and facilitators from a European perspective.

BACKGROUND: With an increasing number of people dying in old age, collaboration between palliative care and geriatric medicine is increasingly being advocated in order to promote better health and health care for the increasing number of older people. The aim of this study is to identify barriers and facilitators and good practice examples of collaboration and integration between palliative care and geriatric medicine from a European perspective. METHODS: Four semi-structured group interviews were undertaken with 32 participants from 18 countries worldwide. Participants were both clinicians (geriatricians, GPs, palliative care specialists) and academic researchers. The interviews were transcribed and independent analyses performed by two researchers who then reached consensus. RESULTS: Limited knowledge and understanding of what the other discipline offers, a lack of common practice and a lack of communication between disciplines and settings were considered as barriers for collaboration between palliative care and geriatric medicine. Multidisciplinary team working, integration, strong leadership and recognition of both disciplines as specialties were considered as facilitators of collaborative working. Whilst there are instances of close clinical working between disciplines, examples of strategic collaboration in education and policy were more limited. CONCLUSIONS: Improving knowledge about its principles and acquainting basic palliative care skills appears mandatory for geriatricians and other health care professionals. In addition, establishing more academic chairs is seen as a priority in order to develop more education and development at the intersection of palliative care and geriatric medicine.

Distinct subcellular localization of the neuronal marker HuC/D reveals hypoxia-induced damage in enteric neurons.

BACKGROUND: Correct neuronal identification is essential to study neurons in health and disease. Although commonly used as pan-neuronal marker, HuC/D's expression pattern varies substantially between healthy and (patho)physiological conditions. This heterogenic labeling has received very little attention. We sought to investigate the subcellular HuC/D localization in enteric neurons in different conditions. METHODS: The localization of neuronal RNA-binding proteins HuC/D was investigated by immunohistochemistry in the mouse myenteric plexus using different toxins and caustic agents. Preparations were also stained with Sox10 and glial fibrillary acidic protein (GFAP) antibodies to assess enteric glial cell appearance. KEY RESULTS: Mechanically induced tissue damage, interference with the respiratory chain and oxygen (O2 ) deprivation increased nuclear HuC/D immunoreactivity. This effect was paralleled by a distortion of the GFAP-labeled glial network along with a loss of Sox10 expression and coincided with the activation of a non-apoptotic genetic program. Chemically induced damage and specific noxious stimuli did not induce a change in HuC/D immunoreactivity, supporting the specific nature of the nuclear HuC/D localization. CONCLUSIONS & INFERENCES: HuC/D is not merely a pan-neuronal marker but its subcellular localization also reflects the condition of a neuron at the time of fixation. The functional meaning of this change in HuC/D localization is not entirely clear, but disturbance in O2 supply in combination with the support of enteric glial cells seems to play a crucial role. The molecular consequence of changes in HuC/D expression needs to be further investigated.

Effect of acute peppermint oil administration on gastric sensorimotor function and nutrient tolerance in health.

BACKGROUND: Menthol reduces intestinal motility in animal studies, an effect that is probably mediated by transient receptor potential channels. Peppermint oil (PO), with menthol as a major constituent, is widely used as a spasmolytic agent in irritable bowel syndrome. In the current study, we investigated the effect of acute PO administration on intragastric pressure (IGP) profiles and gastric sensorimotor functions in health. METHODS: Healthy volunteers underwent IGP measurement before and during continuous intragastric infusion of a nutrient drink (n = 13), and gastric barostat studies (n = 13). A single capsule of PO (182 mg) or placebo was administered during the studies in a randomized controlled crossover design. Throughout the studies, healthy volunteers scored 11 epigastric symptoms on a visual analogue scale (VAS); satiation was scored on a 6-point Likert scale during intragastric infusion. KEY RESULTS: During fasting, IGP and motility index (MI) of the proximal stomach decreased significantly after PO administration compared with placebo (P < 0.0001 and <0.05, respectively). In contrast, during intragastric infusion of the nutrient drink, no significant differences were detected between PO and placebo in IGP profiles, MI, satiation scores, and epigastric symptoms. The maximum infused volume, gastric compliance or sensitivity to balloon distention did not differ between both treatment arms. However, reduced appetite scores were seen during fasting after PO treatment, as compared with placebo (P = 0.01). Postprandial VAS scores were similar between PO and placebo. CONCLUSIONS & INFERENCES: Peppermint oil reduces IGP, proximal phasic contractility, and appetite, with negligible effects on gastric sensitivity, tone, accommodation, and nutrient tolerance in health.

Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells.

BACKGROUND: The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. METHODS: We studied the effect of acetylcholine (ACh), serotonin (5-HT), and adenosine triphosphate (ATP) on intracellular Ca(2+) signaling using aequorin-expressing and Fluo-4 AM-loaded CRL-2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c-Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT-PCR. KEY RESULTS: Apart from ATP, also ACh and 5-HT induced a dose-dependent increase in intracellular Ca(2+) concentration in CRL-2690 cells. Similarly, these neurotransmitters also evoked Ca(2+) transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5-HT induced early gene expression in CRL-2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. CONCLUSIONS & INFERENCES: We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca(2+). On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.

Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge.

Slow changes in [Ca(2+)](i) reflect increased neuronal activity. Our study demonstrates that single-trial fast [Ca(2+)](i) imaging (≥200 Hz sampling rate) revealed peaks each of which are associated with single spike discharge recorded by consecutive voltage-sensitive dye (VSD) imaging in enteric neurones and nerve fibres. Fast [Ca(2+)](i) imaging also revealed subthreshold fast excitatory postsynaptic potentials. Nicotine-evoked [Ca(2+)](i) peaks were reduced by -conotoxin and blocked by ruthenium red or tetrodotoxin. Fast [Ca(2+)](i) imaging can be used to directly record single action potentials in enteric neurones. [Ca(2+)](i) peaks required opening of voltage-gated sodium and calcium channels as well as Ca(2+) release from intracellular stores.

Prolonged IL-1beta exposure alters neurotransmitter and electrically induced Ca(2+) responses in the myenteric plexus.

BACKGROUND Infection and inflammatory diseases of the gut results in profound changes of intestinal motor function. Acute administration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) was shown to have excitatory and neuromodulatory roles in the myenteric plexus. Here we aimed to study the effect of prolonged IL-1beta incubation on the response of myenteric neurones to different stimuli. METHODS Longitudinal muscle myenteric plexus preparations (LMMP's) of the guinea pig jejunum were incubated for 24 h in medium with or without IL-1beta. After loading with Fluo-4, calcium imaging was used to visualize activation of neurones. The response to application of serotonin (5-HT), substance P (SP) and ATP or to electrical fibre tract stimulation (eFTS) was tested. Expression of nNOS, HuD, calbindin and calretinin was compared by immunohistochemistry. KEY RESULTS IL-1beta concentration-dependently influenced the neuronal responsiveness and duration of the [Ca(2+)](i) rises to 5-HT and ATP, while it also affected the Ca(2+)-transient amplitudes induced by 5-HT, ATP and SP. Ca(2+)-transients in response to eFTS were observed in significantly more neurones per ganglion after IL-1beta (10(-10) and 10(-11) mol L(-1)). Peak [Ca(2+)](i) rise after eFTS was concentration-dependently decreased by IL-1beta. The duration of the [Ca(2+)](i) rise after eFTS was prolonged after IL-1beta 10(-12) mol L(-1). IL-1beta (10(-9) mol L(-1)) incubation did not affect the number of nNOS, calretinin and calbindin expressing neurones, nor did it induce neuronal loss (HuD). CONCLUSIONS & INFERENCES In this study, IL-1beta differentially modulates the neuronal response to eFTS and neurotransmitter application in the myenteric plexus of guinea pigs. This cytokine could be implicated in the motility disturbances observed during gastrointestinal inflammation.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH-IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH-IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 microm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP-IR but not nNOS-IR enteric neurons. The KCl-induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 microm), of noradrenaline (1 microm) and of the alpha(2)-adrenoreceptor agonist clonidine (1 microm). Combining pharmacological and calcium imaging studies, we have further shown that L-type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal-regulated kinases were required for the increase in the proportion of TH-IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.

P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine.

There is increasing evidence that adenosine 5'-triphosphate or a related purine plays a crucial role in smooth muscle relaxation and enteric synaptic neurotransmission. Accordingly, the aim of the present work is to investigate the role P2Y(1) receptors in purinergic inhibitory neurotransmission (pig ileum) and enteric neuronal activation in the small intestine (guinea-pig ileum). Using contractility measurements, micro-electrode recordings and Ca(2+) imaging we found that (i) adenosine 5'-Omicron-2-thiodiphosphate (ADPbetaS) (10 micromol L(-1)) caused smooth muscle relaxation and hyperpolarization that was antagonized by MRS2179 (10 micromol L(-1)) a P2Y(1) receptor antagonist and apamin (1 micromol L(-1)); (ii) electrical field stimulation (EFS) caused a non-nitrergic inhibitory junction potential (IJP) and relaxation that was antagonized by MRS2179 (10 micromol L(-1)); (iii) P2Y(1) receptors were immunolocalized in smooth muscle cells and enteric neurons; (i.v.) superfusion of ADPbetaS (1 micromol L(-1)) induced Ca(2+) transients in myenteric neurons that were inhibited by MRS2179 (1 micromol L(-1)), but not by tetrodotoxin (1 micromol L(-1)); and (v) EFS induced calcium transients were partially inhibited by MRS2179 (1 micromol L(-1)). We conclude that in the small intestine purinergic neuromuscular transmission responsible for the IJP and non-nitrergic relaxation is mediated by P2Y(1) receptors located in smooth muscle cells. Functional P2Y(1) receptors are also present in guinea-pig myenteric neurons. Therefore, P2Y(1) receptors might be an important pharmacological target to modulate gastrointestinal functions.

The effect of nitric oxide donors on nitric oxide synthase-expressing myenteric neurones in culture.

Previously, we demonstrated that intestinal inflammation leads to a postinflammatory loss of nitric oxide synthase (NOS)-expressing myenteric neurones and motility disturbances. Here, we investigated whether high NO concentrations could be responsible for the decrease in NOS neurones. Myenteric neurone cultures, prepared from guinea-pig small intestine, were incubated with NO donors [sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1)]. After fixation, NOS neurones were identified by NADPH diaphorase staining and neurone-specific enolase (NSE)-positive neuronal content was assessed with an enzyme-linked immunosorbent assay (ELISA)-based method. Twenty-four hours incubation with SIN-1 (10(-3) mol L(-1)) or SNP (10(-4) mol L(-1) or higher) reduced the number of NADPH diaphorase-positive neurones. SNP incubation did not affect the NSE-positive neuronal content. Shorter incubations (SNP: 4 and 12 h) had no significant effect. The SNP-induced reduction was reversed by glutathione (GSH), but not by NO- or O-scavengers, whereas GSH depletion enhanced the decrease. The NO-dependent guanylate cyclase-blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) did not affect the SNP effect. This reduction can be explained by either specific apoptosis of NOS neurones or downregulation of NOS activity. However, TdT-mediated X-dUTP nick end labelling (TUNEL stainings argue in favour of the latter. In conclusion, the NO donor SNP decreases the number of NOS-expressing myenteric neurones time and concentration dependently, without affecting the amount of neuronal material. Glutathione plays an important protective role.

Role of endogenous ghrelin in the hyperphagia of mice with streptozotocin-induced diabetes.

Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin(-/-)) and control wild-type (ghrelin(+/+)) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin(-/-) mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000-1200 h) in ghrelin(+/+) mice was abolished in mutant mice. Diabetic ghrelin(-/-) mice lost 12.4% more body weight than ghrelin(+/+) mice. In diabetic ghrelin(+/+) mice, but not in ghrelin(-/-) mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and alpha-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced alpha-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.

Neurochemical coding of myenteric neurons in the guinea-pig antrum.

Electrophysiological studies of myenteric neurons in the guinea-pig antrum suggest that different neuroactive compounds are involved in synaptic transmission. It is not known what neurotransmitters and neuropeptides are present and to what extent they colocalize. Immunohistochemical stainings were performed on whole-mount preparations of the guinea-pig antrum. Immunoreactivity for neuron-specific enolase was used as a general marker and was set at 100%. There was no overlap between cholinergic and nitrergic neurons, resulting in two separate subpopulations. The presence of choline acetyltransferase immunoreactivity was used to identify the cholinergic subset, which accounted for 56% of the cells. Immunoreactivity for nitric oxide synthase, on the other hand, was displayed in 40.7% of the neurons. Substance-P immunoreactivity was present in 37.4% of the cells and vasoactive intestinal peptide and neuropeptide Y in 21.7% and 28.6%, respectively. Small subsets of neurons had immunoreactivity for serotonin (3.9%), calretinin (6.8%) and calbindin (0.5%). Colocalization studies revealed several subgroups of neurons, containing one or more of the screened markers. Though some similarity is found in the chemical coding of the antrum compared to that of the small intestine and the corpus, remarkable differences can be seen in the occurrence of some subpopulations. Cholinergic neurons are not as predominant as in other parts of the gut, serotonin presence is doubled and some vasointestinal-peptide-positive neurons express substance P. These differences might reflect the highly specialized function of the antrum; however, the exact role of these classes remains to be established.