Anti-TNFα therapy in IBD alters brain activity reflecting visceral sensory function and cognitive-affective biases.

BACKGROUND: In inflammatory bowel disease (IBD), immune activation with increased circulating TNF-α is linked to the intensity of gastrointestinal symptoms and depression or anxiety. A central feature of depression is cognitive biases linked to negative attributions about self, the world and the future. We aimed to assess the effects of anti-TNFα therapy on the central processing of self-attribution biases and visceral afferent information in patients with Crohn's disease. METHODS: We examined 9 patients with Crohn's disease (age 26.1±10.6. yrs, 5 female, 5 ileocolonic, 2 colonic and 2 ileal disease) during chronic anti-TNFα therapy (5 adalimumab, 4 infliximab). Patients were studied twice in randomized order before and after anti-TNFα administration. On each occasion patients underwent functional magnetic resonance imaging (fMRI) of the brain during a test of implicit attribution biases regarding sickness/health and undertook a standardized nutrient challenge. RESULTS: Following anti-TNFα treatment, ratings of 'fullness' following nutrient challenge reduced compared to pre-treatment ratings (p<0.05). Reaction times revealed improved processing of self-related and positive health words, consistent with improved implicit sense of wellbeing that correlated with improvements in sensory function after treatment (r = 0.67, p<0.05). Treatment-associated improvements in implicit processing were mirrored by alterations of prefrontal, amygdala, posterior cingulate and visual regions. Between patients, the degree of functional amygdala change was additionally explained by individual differences in attention regulation and body awareness rankings. CONCLUSION: In patients with Crohn's disease, anti-TNFα administration reduces visceral sensitivity and improves implicit cognitive-affective biases linked to alterations in limbic (amygdala) function.

Dynasore blocks evoked release while augmenting spontaneous synaptic transmission from primary visceral afferents.

The recycling of vesicle membrane fused during exocytosis is essential to maintaining neurotransmission. The GTPase dynamin is involved in pinching off membrane to complete endocytosis and can be inhibited by dynasore resulting in activity-dependent depletion of release-competent synaptic vesicles. In rat brainstem slices, we examined the effects of dynasore on three different modes of glutamate release-spontaneous, evoked, and asynchronous release-at solitary tract (ST) inputs to neurons in the nucleus of the solitary tract (NTS). Intermittent bursts of stimuli to the ST interspersed with pauses in stimulation allowed examination of these three modes in each neuron continuously. Application of 100 µM dynasore rapidly increased the spontaneous EPSC (sEPSC) frequency which was followed by inhibition of both ST-evoked EPSCs (ST-EPSC) as well as asynchronous EPSCs. The onset of ST-EPSC failures was not accompanied by amplitude reduction-a pattern more consistent with conduction block than reduced probability of vesicle release. Neither result suggested that dynasore interrupted endocytosis. The dynasore response profile resembled intense presynaptic TRPV1 activation. The TRPV1 antagonist capsazepine failed to prevent dynasore increases in sEPSC frequency but did prevent the block of the ST-EPSC. In contrast, the TRPV1 antagonist JNJ 17203212 prevented both actions of dynasore in neurons with TRPV1-expressing ST inputs. In a neuron lacking TRPV1-expressing ST inputs, however, dynasore promptly increased sEPSC rate followed by block of ST-evoked EPSCs. Together our results suggest that dynasore actions on ST-NTS transmission are TRPV1-independent and changes in glutamatergic transmission are not consistent with changes in vesicle recycling and endocytosis.

Effects of methylmercury on spinal cord afferents and efferents-A review.

Methylmercury (MeHg) is an environmental neurotoxicant of public health concern. It readily accumulates in exposed humans, primarily in neuronal tissue. Exposure to MeHg, either acutely or chronically, causes severe neuronal dysfunction in the central nervous system and spinal neurons; dysfunction of susceptible neuronal populations results in neurodegeneration, at least in part through Ca2+-mediated pathways. Biochemical and morphologic changes in peripheral neurons precede those in central brain regions, despite the fact that MeHg readily crosses the blood-brain barrier. Consequently, it is suggested that unique characteristics of spinal cord afferents and efferents could heighten their susceptibility to MeHg toxicity. Transient receptor potential (TRP) ion channels are a class of Ca2+-permeable cation channels that are highly expressed in spinal afferents, among other sensory and visceral organs. These channels can be activated in numerous ways, including directly via chemical irritants or indirectly via Ca2+ release from intracellular storage organelles. Early studies demonstrated that MeHg interacts with heterologous TRP channels, though definitive mechanisms of MeHg toxicity on sensory neurons may involve more complex interaction with, and among, differentially-expressed TRP populations. In spinal efferents, glutamate receptors of the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and possibly kainic acid (KA) classes are thought to play a major role in MeHg-induced neurotoxicity. Specifically, the Ca2+-permeable AMPA receptors, which are abundant in motor neurons, have been identified as being involved in MeHg-induced neurotoxicity. In this review, we will describe the mechanisms that could contribute to MeHg-induced spinal cord afferent and efferent neuronal degeneration, including the possible mediators, such as uniquely expressed Ca2+-permeable ion channels.

Experimental colitis in rats induces de novo synthesis of cytokines at distant intestinal sites: role of capsaicin-sensitive primary afferent fibers.

Increased levels of pro- and anti-inflammatory cytokines were observed in various segments of histologically-intact small intestine in animal models of acute and chronic colitis. Whether these cytokines are produced locally or spread from the inflamed colon is not known. In addition, the role of gut innervation in this upregulation is not fully understood. To examine whether cytokines are produced de novo in the small intestine in two rat models of colitis; and to investigate the role of capsaicin-sensitive primary afferents in the synthesis of these inflammatory cytokines. Colitis was induced by rectal instillation of iodoacetamide (IA) or trinitrobenzene sulphonic acid (TNBS) in adult Sprague-Dawley rats. Using reverse transcriptase (RT) and real-time PCR, TNF-α, and IL-10 mRNA expression was measured in mucosal scrapings of the duodenum, jejunum, ileum and colon at different time intervals after induction of colitis. Capsaicin-sensitive primary afferents (CSPA) were ablated using subcutaneous injections of capsaicin at time 0, 8 and 32 h, and the experiment was repeated at specific time intervals to detect any effect on cytokines expression. TNF-α mRNA expression increased by 3-40 times in the different intestinal segments (p<0.05 to p<0.001), 48h after IA-induced colitis. CSPA ablation completely inhibited this upregulation in the small intestine, but not in the colon. Similar results were obtained in TNBS-induced colitis at 24 h. Intestinal IL-10 mRNA expression significantly decreased at 12 h and then increased by 6-43 times (p<0.05 to p<0.001) 48h after IA administration. This increase was abolished in rats subjected to CSPA ablation except in the colon, where IL-10 further increased by twice (p<0.05). In the TNBS group, there was 4-12- and 4-7-fold increases in small intestinal IL-10 mRNA expression at 1 and 21 days after colitis induction, respectively (both p<0.01). This increase was not observed in rats pretreated with capsaicin. Capsaicin-treated and untreated rats had comparable visual ulcer scores after colitis induction. Inflammatory cytokines are produced de novo in distant intestinal segments in colitis. CSPA fibers play a key role in the upregulation of this synthesis.

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease-derived stimuli.

Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9(-/-) mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus-response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9(-/-) afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9(-/-) mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.

Shugan-decoction relieves visceral hyperalgesia and reduces TRPV1 and SP colon expression.

AIM: To evaluate the therapeutic effect of Shugan-decoction (SGD) on visceral hyperalgesia and colon gene expressions using a rat model. METHODS: Ninety-six adult male Wistar rats were randomized into six equal groups for assessment of SGD effects on psychological stress-induced changes using the classic water avoidance stress (WAS) test. Untreated model rats were exposed to chronic (1 h/d for 10 d consecutive) WAS conditions; experimental treatment model rats were administered with intragastric SGD at 1 h before WAS on consecutive days 4-10 (low-dose: 0.1 g/mL; mid-dose: 0.2 g/mL; high-dose: 0.4 g/mL); control treatment model rats were similarly administered with the irritable bowel syndrome drug, dicetel (0.0042 g/mL); untreated normal control rats received no drug and were not subjected to the WAS test. At the end of the 10-d WAS testing period, a semi-quantitative measurement of visceral sensitivity was made by assessing the abdominal withdrawal reflex (AWR) to colorectal balloon-induced distension (at 5 mmHg increments) to determine the pain pressure threshold (PPT, evidenced by pain behavior). Subsequently, the animals were sacrificed and colonic tissues collected for assessment of changes in expressions of proteins related to visceral hypersensitivity (transient receptor potential vanilloid 1, TRPV1) and sustained visceral hyperalgesia (substance P, SP) by immunohistochemistry and real-time polymerase chain reaction. Inter-group differences were assessed by paired t test or repeated measures analysis of variance. RESULTS: The WAS test successfully induced visceral hypersensitivity, as evidenced by a significantly reduced AWR pressure in the untreated model group as compared to the untreated normal control group (190.4 ± 3.48 mmHg vs 224.0 ± 4.99 mmHg, P < 0.001). SGD treatments at mid-dose and high-dose and the dicetel treatment significantly increased the WAS-reduced PPT (212.5 ± 2.54, 216.5 ± 3.50 and 217.7 ± 2.83 mmHg respectively, all P < 0.001); however, the low-dose SGD treatment produced no significant effect on the WAS-reduced PPT (198.3 ± 1.78 mmHg, P > 0.05). These trends corresponded to the differential expressions observed for both TRPV1 protein (mid-dose: 1.64 ± 0.08 and high-dose: 1.69 ± 0.12 vs untreated model: 3.65 ± 0.32, P < 0.001) and mRNA (0.44 ± 0.16 and 0.15 ± 0.03 vs 1.39 ± 0.15, P < 0.001) and SP protein (0.99 ± 0.20 and 1.03 ± 0.23 vs 2.03 ± 0.12, P < 0.01) and mRNA (1.64 ± 0.19 and 1.32 ± 0.14 vs 2.60 ± 0.33, P < 0.05). These differential expressions of TRPV1 and SP related to mid- and high-dose SGD treatments were statistically similar to the changes induced by dicetel treatment. No signs of overt damage to the rat system were observed for any of the SGD dosages. CONCLUSION: Shugan-decoction can reduce chronic stress-induced visceral hypersensitivity in rats, and the regulatory mechanism may involve mediating the expressions of TRPV1 and SP in colon tissues.

Pharmacological pain management in chronic pancreatitis.

Intense abdominal pain is a prominent feature of chronic pancreatitis and its treatment remains a major clinical challenge. Basic studies of pancreatic nerves and experimental human pain research have provided evidence that pain processing is abnormal in these patients and in many cases resembles that seen in neuropathic and chronic pain disorders. An important ultimate outcome of such aberrant pain processing is that once the disease has advanced and the pathophysiological processes are firmly established, the generation of pain can become self-perpetuating and independent of the initial peripheral nociceptive drive. Consequently, the management of pain by traditional methods based on nociceptive deafferentation (e.g., surgery and visceral nerve blockade) becomes difficult and often ineffective. This novel and improved understanding of pain aetiology requires a paradigm shift in pain management of chronic pancreatitis. Modern mechanism based pain treatments taking into account altered pain processing are likely to increasingly replace invasive therapies targeting the nociceptive source, which should be reserved for special and carefully selected cases. In this review, we offer an overview of the current available pharmacological options for pain management in chronic pancreatitis. In addition, future options for pain management are discussed with special emphasis on personalized pain medicine and multidisciplinarity.

Mast cells and the cyclooxygenase pathway mediate colonic afferent nerve sensitization in a murine colitis model.

INTRODUCTION: Intestinal inflammation alters colonic afferent nerve sensitivity which may contribute to patients' perception of abdominal discomfort. We aimed to explore whether mast cells and the cyclooxygenase pathway are involved in altered afferent nerve sensitivity during colitis. METHODS: C57Bl6 mice received 3% dextran-sulfate sodium (DSS) in drinking water for 7 days to induce colitis. Control animals received regular water. On day 8 inflammation was assessed in the proximal colon by morphology and histology. Extracellular afferent nerve discharge was recorded from the mesenteric nerve of a 2 cm colonic segment. Subgroups were treated in vitro with the mast cell stabilizer doxantrazole (10⁻4M) or the cyclooxygenase inhibitor naproxen (10⁻5M). RESULTS: DSS colitis resulted in morphological and histological signs of inflammation. At baseline, peak firing was 11±2 imp s⁻¹ in colitis segments and 5±1 imp s⁻¹ in uninflamed control segments (p<0.05; mean ± SEM; each n=6). In colitis segments, afferent nerve discharge to bradykinin (0.5 µM) was increased to 47±7 compared to 23±6 imp s⁻¹ in recordings from non-inflamed control tissue (p<0.05). Mechanosensitivity during luminal ramp distension (0-80 cm H2O) was increased reaching 24±5 imp s⁻¹ at 80 cm H2O during colitis compared to 14±2 in non-inflamed controls (p<0.05). Doxantrazole or naproxen reduced afferent discharge to bradykinin and luminal ramp distension in colitis segments to control levels. CONCLUSION: Intestinal inflammation sensitizes mesenteric afferent nerve fibers to bradykinin and mechanical stimuli. The underlying mechanism responsible for this sensitization seems to involve mast cells and prostaglandins.

The mast cell degranulator compound 48/80 directly activates neurons.

BACKGROUND: Compound 48/80 is widely used in animal and tissue models as a "selective" mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. METHODOLOGY/PRINCIPAL FINDINGS: We used in vivo recordings from extrinsic intestinal afferents together with Ca(++) imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca(++) and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca(++) transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H(1) and H(2) antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca(++) transients in mast cell-free enteric neuron cultures. CONCLUSIONS/SIGNIFICANCE: The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.

Sex differences in spinal processing of transient and inflammatory colorectal stimuli in the rat.

Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil-treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats.

[Adrenaline potentiates antiepileptic but not sedative action of diazepam in rats].

Intramuscular (i.m.) administration ofdiazepam in a dose of 10 mg/kg and adrenaline in a dose of 0.2 mg/kg prevents generalized clonic-tonic pentylenetetrazol (PTZ) seizures in 75-80 % of rats, but only in 35-40 % of rats it prevents local clonic PTZ seizures. In the above mentioned dose, diazepam causes a strong sedation, but adrenaline does not cause a sedative effects. The combined administration of diazepam and adrenaline in threshold independently ineffective doses prevents both clonic-tonic and clonic PTZ seizures in 80 % of rats without a sedation development. The basis for mechanism of potentiation of anticonvulsant action of diazepam is the stimulation of gastric mucosa afferents by adrenaline.

Degeneration of sensory afferent nerves enhances pulmonary inflammatory alterations in acute myocardial infarction in rats.

BACKGROUND: Evidence suggests proinflammatory changes in the lungs during acute myocardial infarction and a participation of neural mechanisms and substance P in the pathology. This study was undertaken to investigate the role and the mechanisms by which sensory afferent degeneration at neonatal stages exacerbates the pulmonary inflammatory responses to acute myocardial infarction in the adult rats. METHODS: The degeneration of capsaicin-sensitive afferent nerves was induced by administration of capsaicin to neonatal rats. The pulmonary inflammatory changes following coronary artery occlusion (CAO) were assessed by the analysis of the infiltration of neutrophils and tissue morphology in the lungs. RESULTS: Significant increases in the pulmonary infiltration of neutrophils, up to 240% and 218% of the sham controls at 3 and 6 h, respectively, after CAO (P<.05) and marked pulmonary edema were observed. Degeneration of capsaicin-sensitive afferent nerves or antagonism of endogenous neurokinin (NK)-1 receptor exacerbated the pulmonary infiltration of neutrophils (up to 214% and 254% of the controls, respectively) and pulmonary tissue edema following the CAO. CONCLUSION: The findings indicate that degeneration of sensory afferent nerves enhances the pulmonary inflammatory changes in acute myocardial infarction, in which the endogenous NK may play a role.

Effects of gastric distension and infusion of umami and bitter taste stimuli on vagal afferent activity.

Until recently, sensory nerve pathways from the stomach to the brain were thought to detect distension and play little role in nutritional signaling. Newer data have challenged this view, including reports on the presence of taste receptors in the gastrointestinal lumen and the stimulation of multi-unit vagal afferent activity by glutamate infusions into the stomach. However, assessing these chemosensory effects is difficult because gastric infusions typically evoke a distension-related vagal afferent response. In the current study, we recorded gastric vagal afferent activity in the rat to investigate the possibility that umami (glutamate, 150 mM) and bitter (denatonium, 10 mM) responses could be dissociated from distension responses by adjusting the infusion rate and opening or closing the drainage port in the stomach. Slow infusions of saline (5 ml over 2 min, open port) produced no significant effects on vagal activity. Using the same infusion rate, glutamate or denatonium solutions produced little or no effects on vagal afferent activity. In an attempt to reproduce a prior report that showed distention and glutamate responses, we produced a distension response by closing the exit port. Under this condition, response to the infusion of glutamate or denatonium was similar to saline. In summary, we found little or no effect of gastric infusion of glutamate or denatonium on gastric vagal afferent activity that could be distinguished from distension responses. The current results suggest that sensitivity to umami or bitter stimuli is not a common property of gastric vagal afferent fibers.

TRPV1 marks synaptic segregation of multiple convergent afferents at the rat medial solitary tract nucleus.

TRPV1 receptors are expressed on most but not all central terminals of cranial visceral afferents in the caudal solitary tract nucleus (NTS). TRPV1 is associated with unmyelinated C-fiber afferents. Both TRPV1+ and TRPV1- afferents enter NTS but their precise organization remains poorly understood. In horizontal brainstem slices, we activated solitary tract (ST) afferents and recorded ST-evoked glutamatergic excitatory synaptic currents (ST-EPSCs) under whole cell voltage clamp conditions from neurons of the medial subnucleus. Electrical shocks to the ST produced fixed latency EPSCs (jitter<200 µs) that identified direct ST afferent innervation. Graded increases in shock intensity often recruited more than one ST afferent and ST-EPSCs had consistent threshold intensity, latency to onset, and unique EPSC waveforms that characterized each unitary ST afferent contact. The TRPV1 agonist capsaicin (100 nM) blocked the evoked TRPV1+ ST-EPSCs and defined them as either TRPV1+ or TRPV1- inputs. No partial responses to capsaicin were observed so that in NTS neurons that received one or multiple (2-5) direct ST afferent inputs--all were either blocked by capsaicin or were unaltered. Since TRPV1 mediates asynchronous release following TRPV1+ ST-evoked EPSCs, we likewise found that recruiting more than one ST afferent further augmented the asynchronous response and was eliminated by capsaicin. Thus, TRPV1+ and TRPV1- afferents are completely segregated to separate NTS neurons. As a result, the TRPV1 receptor augments glutamate release only within unmyelinated afferent pathways in caudal medial NTS and our work indicates a complete separation of C-type from A-type afferent information at these first central neurons.

A novel role for TRPM8 in visceral afferent function.

Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperatures and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of bowel hypersensitivity; however, the underlying mechanisms of action are unclear. Here we determined the role of TRPM8 in colonic sensory pathways. Laser capture microdissection, quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and retrograde tracing were used to localise TRPM8 to colonic primary afferent neurons. In vitro extracellular single-fibre afferent recordings were used to determine the effect of TRPM8 channel activation on the chemosensory and mechanosensory function of colonic high-threshold afferent fibres. TRPM8 mRNA was present in colonic DRG neurons, whereas TRPM8 protein was present on nerve fibres throughout the wall of the colon. A subpopulation (24%, n=58) of splanchnic serosal and mesenteric afferents tested responded directly to icilin (5 µmol/L). Subsequently, icilin significantly desensitised afferents to mechanical stimulation (P<.0001; n=37). Of the splanchnic afferents responding to icilin, 21 (33%) also responded directly to the TRPV1 agonist capsaicin (3 µmol/L), and icilin reduced the direct chemosensory response to capsaicin. Icilin also prevented mechanosensory desensitization and sensitization induced by capsaicin and the TRPA1 agonist AITC (40 µmol/L), respectively. TRPM8 is present on a select population of colonic high threshold sensory neurons, which may also co-express TRPV1. TRPM8 couples to TRPV1 and TRPA1 to inhibit their downstream chemosensory and mechanosensory actions.

Intestinal serotonin release, sensory neuron activation, and abdominal pain in irritable bowel syndrome.

OBJECTIVES: Serotonin (5-hydroxytryptamine, 5-HT) metabolism may be altered in gut disorders, including in the irritable bowel syndrome (IBS). We assessed in patients with IBS vs. healthy controls (HCs) the number of colonic 5-HT-positive cells; the amount of mucosal 5-HT release; their correlation with mast cell counts and mediator release, as well as IBS symptoms; and the effects of mucosal 5-HT on electrophysiological responses in vitro. METHODS: We enrolled 25 Rome II IBS patients and 12 HCs. IBS symptom severity and frequency were graded 0-4. 5-HT-positive enterochromaffin cells and tryptase-positive mast cells were assessed with quantitative immunohistochemistry on colonic biopsies. Mucosal 5-HT and mast cell mediators were assessed by high-performance liquid chromatography or immunoenzymatic assay, respectively. The impact of mucosal 5-HT on electrophysiological activity of rat mesenteric afferent nerves was evaluated in vitro. RESULTS: Compared with HCs, patients with IBS showed a significant increase in 5-HT-positive cell counts (0.37 ± 0.16% vs. 0.56 ± 0.26%; P=0.039), which was significantly greater in patients with diarrhea-predominant IBS vs. constipation-predominant IBS (P=0.035). Compared with HCs, 5-HT release in patients with IBS was 10-fold significantly increased (P < 0.001), irrespective of bowel habit, and was correlated with mast cell counts. A significant correlation was found between the mucosal 5-HT release and the severity of abdominal pain (r(s)=0.582, P=0.047). The area under the curve, but not peak sensory afferent discharge evoked by IBS samples in rat jejunum, was significantly inhibited by the 5-HT3 receptor antagonist granisetron (P<0.005). CONCLUSIONS: In patients with IBS, 5-HT spontaneous release was significantly increased irrespective of bowel habit and correlated with mast cell counts and the severity of abdominal pain. Our results suggest that increased 5-HT release contributes to development of abdominal pain in IBS, probably through mucosal immune activation.

Modulation of the assay system for the sensory integration of 2 sensory stimuli that inhibit each other in nematode Caenorhabditis elegans.

OBJECTIVE: To perform the modulation of an assay system for the sensory integration of 2 sensory stimuli that inhibit each other. METHODS: The assay system for assessing the integrative response to 2 reciprocally-inhibitory sensory stimuli was modulated by changing the metal ion barrier. Moreover, the hen-1, ttx-3 and casy-1 mutants having known defects in integrative response were used to evaluate the modulated assay systems. Based on the examined assay systems, new genes possibly involved in the sensory integration control were identified. RESULTS: In the presence of different metal ion barriers and diacetyl, locomotion behaviors, basic movements, pan-neuronal, cholinergic and GABAergic neuronal GFP expressions, neuronal development, structures of sensory neurons and interneurons, and stress response of nematodes in different regions of examined assay systems were normal, and chemotaxis toward different concentrations of diacetyl and avoidance of different concentrations of metal ions were inhibited. In the first group, most of the nematodes moved to diacetyl by crossing the barrier of Fe(2+), Zn(2+), or Mn(2+). In the second group, almost half of the nematodes moved to diacetyl by crossing the barrier of Ag(+), Cu(2+), Cr(2+), or Cd(2+). In the third group, only a small number of nematodes moved to diacetyl by crossing the barrier of Pb(2+) or Hg(2+). Moreover, when nematodes encountered different metal ion barriers during migration toward diacetyl, the percentage of nematodes moving back and then turning and that of nematodes moving straight to diacetyl were very different. With the aid of examined assay systems, it was found that mutations of fsn-1 that encodes a F-box protein, and its target scd-2 that encodes a receptor tyrosine kinase, caused severe defects in integrative response, and the sensory integration defects of fsn-1 mutants were obviously inhibited by scd-2 mutation. CONCLUSION: Based on the nematode behaviors in examined assay systems, 3 groups of assay systems were obtained. The first group may be helpful in evaluating or identifying the very subtle deficits in sensory integration, and the third group may be useful for the final confirmation of sensory integration defects of mutants identified in the first or the second group of assay systems. Furthermore, the important association of sensory integration regulation with stabilization or destabilization of synaptic differentiation may exist in C. elegans.

Human visceral afferent recordings: preliminary report.

BACKGROUND: Conditions characterised by chronic visceral pain represent a significant healthcare burden with limited treatment options. While animal models have provided insights into potential mechanisms of visceral nociception and identified candidate drug targets, these have not translated into successful treatments in humans. OBJECTIVE: To develop an in vitro afferent nerve preparation using surgically excised freshly isolated human colon and vermiform appendix-mesentery tissues. METHODS: Non-inflamed appendix (n=18) and colon (n=9) were collected from patients undergoing right and left hemicolectomy. Electrophysiological recordings were made from mesenteric nerves and the tissue stimulated chemically and mechanically. RESULTS: Ongoing neuronal activity was sparse and where units occurred peak firing rates were: colon (2.0±0.4 spikes/s, n=4) and appendix (2.4±0.6 spikes/s, n=9). Afferent nerves innervating the appendix responded with a significant increase in activity following stimulation with inflammatory mediators (73±10.6 vs 3.0±0.3 spikes/s, n=6, p<0.001, inflammatory mediator vs baseline) and capsaicin (63±15.8 vs 2±0.3 spikes/s, n=3, p<0.001, capsaicin vs buffer). Afferent nerves innervating the colon responded with increased activity to blunt probing of the serosal surface. CONCLUSIONS: This first-in-human study demonstrates afferent nerve recordings from human gut tissue ex vivo and shows that tissue may be stimulated both chemically and mechanically to study neuronal responses. Collectively, the results provide preliminary evidence to validate this in vitro human tissue model as one that may aid future disease mechanistic studies and candidate drug testing.

Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice.

Inflammation or pain during neonatal development can result in long-term structural and functional alterations of nociceptive pathways, ultimately altering pain perception in adulthood. We have developed a mouse model of neonatal colon irritation (NCI) to investigate the plasticity of pain processing within the viscerosensory system. Mouse pups received an intracolonic administration of 2% mustard oil (MO) on postnatal days 8 and 10. Distal colons were processed at subsequent timepoints for myeloperoxidase (MPO) activity and growth factor expression. Adult mice were assessed for visceral hypersensitivity by measuring the visceromotor response during colorectal distension. Dorsal root ganglion (DRG) neurons from adult mice were retrogradely labeled from the distal colon and calcium imaging was used to measure transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) responses to acute application of capsaicin and MO, respectively. Despite the absence of inflammation (as indicated by MPO activity), neonatal exposure to intracolonic MO transiently maintained a higher expression level of growth factor messenger RNA (mRNA). Adult NCI mice displayed significant visceral hypersensitivity, as well as increased sensitivity to mechanical stimulation of the hindpaw, compared to control mice. The percentage of TRPA1-expressing colon afferents was significantly increased in NCI mice, however they displayed no increase in the percentage of TRPV1-immunopositive or capsaicin-sensitive colon DRG neurons. These results suggest that early neonatal colon injury results in a long-lasting visceral hypersensitivity, possibly driven by an early increase in growth factor expression and maintained by permanent changes in TRPA1 function.