Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Development of the intrinsic innervation of the small bowel mucosa and villi.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth.

Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice.

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.

Ebselen prevents cigarette smoke-induced gastrointestinal dysfunction in mice.

Gastrointestinal (GI) dysfunction is a common comorbidity of chronic obstructive pulmonary disease (COPD) for which a major cause is cigarette smoking (CS). The underlying mechanisms and precise effects of CS on gut contractility, however, are not fully characterised. Therefore, the aim of the present study was to investigate whether CS impacts GI function and structure in a mouse model of CS-induced COPD. We also aimed to investigate GI function in the presence of ebselen, an antioxidant that has shown beneficial effects on lung inflammation resulting from CS exposure. Mice were exposed to CS for 2 or 6 months. GI structure was analysed by histology and immunofluorescence. After 2 months of CS exposure, ex vivo gut motility was analysed using video-imaging techniques to examine changes in colonic migrating motor complexes (CMMCs). CS decreased colon length in mice. Mice exposed to CS for 2 months had a higher frequency of CMMCs and a reduced resting colonic diameter but no change in enteric neuron numbers. Ten days cessation after 2 months CS reversed CMMC frequency changes but not the reduced colonic diameter phenotype. Ebselen treatment reversed the CS-induced reduction in colonic diameter. After 6 months CS, the number of myenteric nitric-oxide producing neurons was significantly reduced. This is the first evidence of colonic dysmotility in a mouse model of CS-induced COPD. Dysmotility after 2 months CS is not due to altered neuron numbers; however, prolonged CS-exposure significantly reduced enteric neuron numbers in mice. Further research is needed to assess potential therapeutic applications of ebselen in GI dysfunction in COPD.

Blockage of the P2X7 Receptor Attenuates Harmful Changes Produced by Ischemia and Reperfusion in the Myenteric Plexus.

INTRODUCTION: Our work analyzed the effects of a P2X7 receptor antagonist, Brilliant Blue G (BBG), on rat ileum myenteric plexus following ischemia and reperfusion (ISR) induced by 45 min of ileal artery occlusion with an atraumatic vascular clamp with 24 h (ISR 24-h group) or 14 d of reperfusion (ISR 14-d group). MATERIAL AND METHODS: Either BBG (50 mg/kg or 100 mg/kg, BBG50 or BBG100 groups) or saline (vehicle) was administered subcutaneously 1 h after ischemia in the ISR 24-h group or once daily for the 5 d after ischemia in the ISR 14-d group (n = 5 per group). We evaluated the neuronal density and profile area by examining the number of neutrophils in the intestinal layers, protein expression levels of the P2X7 receptor, intestinal motility and immunoreactivity for the P2X7 receptor, nitric oxide synthase, neurofilament-200, and choline acetyl transferase in myenteric neurons. RESULTS: The neuronal density and profile area were restored by BBG following ISR. The ischemic groups showed alterations in P2X7 receptor protein expression and the number of neutrophils in the intestine and decreased intestinal motility, all of which were recovered by BBG treatment. CONCLUSION: We concluded that ISR morphologically and functionally affected the intestine and that its effects were reversed by BBG treatment, suggesting the P2X7 receptor as a therapeutic target.

Effect of acetylsalicylic acid on total myenteric neurons in mice experimentally infected with Trypanosoma cruzi.

We investigated the effects of acetylsalicylic acid (ASA) on the total myenteric neuronal population in the descending colon in Trypanosoma cruzi-infected mice. Thirty-five male Swiss mice, 60 days old, were divided into a control group (C group), control group treated with ASA (CA group), infected group (I group), and infected group treated with ASA (IA group). A total of 1300 trypomastigotes of the Y strain of T. cruzi were intraperitoneally inoculated in the IA and I groups. The CA and IA groups were treated with ASA intraperitoneally. At 75 days post-infection (dpi), all of the animals were sacrificed. Neurons in the colon were stained with Giemsa, quantified, and measured. No difference in the course of infection was observed between the IA and I groups, reflected by the parasitemia curve. Acetylsalicylic acid treatment in the CA and IA groups did not alter the total number of myenteric neurons compared with the C and I groups. The CA and IA groups exhibited an increase in the nuclear area, cytoplasmic area, and neuronal body area compared with the C and I groups. Future studies should elucidate the mechanism of action of ASA against Chagas' disease in the chronic phase.

Influence of Acrylamide Administration on the Neurochemical Characteristics of Enteric Nervous System (ENS) Neurons in the Porcine Duodenum.

The digestive tract, especially the small intestine, is one of the main routes of acrylamide absorption and is therefore highly exposed to the toxic effect of acrylamide contained in food. The aim of this experiment was to elucidate the effect of low (tolerable daily intake-TDI) and high (ten times higher than TDI) doses of acrylamide on the neurochemical phenotype of duodenal enteric nervous system (ENS) neurons using the pig as an animal model. The experiment was performed on 15 immature gilts of the Danish Landrace assigned to three experimental groups: control (C) group-pigs administered empty gelatine capsules, low dose (LD) group-pigs administered capsules with acrylamide at the TDI dose (0.5 µg/kg body weight (b.w.)/day), and the high dose (HD) group-pigs administered capsules with acrylamide at a ten times higher dose than the TDI (5 µg/kg b.w./day) with a morning feeding for 4 weeks. Administration of acrylamide, even in a low (TDI) dose, led to an increase in the percentage of enteric neurons immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), neuronal nitric oxide synthase (nNOS), and vesicular acetylcholine transporter (VACHT) in the porcine duodenum. The severity of the changes clearly depended on the dose of acrylamide and the examined plexus. The obtained results suggest the participation of these neuroactive substances in acrylamide-inducted plasticity and the protection of ENS neurons, which may be an important line of defence from the harmful action of acrylamide.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 µM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

VIP is involved in peripheral CRF-induced stimulation of propulsive colonic motor function and diarrhea in male rats.

We investigated whether vasoactive intestinal peptide (VIP) and/or prostaglandins contribute to peripheral corticotropin-releasing factor (CRF)-induced CRF1 receptor-mediated stimulation of colonic motor function and diarrhea in rats. The VIP antagonist, [4Cl-D-Phe6, Leu17]VIP injected intraperitoneally completely prevented CRF (10 µg/kg ip)-induced fecal output and diarrhea occurring within the first hour after injection, whereas pretreatment with the prostaglandins synthesis inhibitor, indomethacin, had no effect. In submucosal plexus neurons, CRF induced significant c-Fos expression most prominently in the terminal ileum compared with duodenum and jejunum, whereas no c-Fos was observed in the proximal colon. c-Fos expression in ileal submucosa was colocalized in 93.4% of VIP-positive neurons and 31.1% of non-VIP-labeled neurons. CRF1 receptor immunoreactivity was found on the VIP neurons. In myenteric neurons, CRF induced only a few c-Fos-positive neurons in the ileum and a robust expression in the proximal colon (17.5 ± 2.4 vs. 0.4 ± 0.3 cells/ganglion in vehicle). The VIP antagonist prevented intraperitoneal CRF-induced c-Fos induction in the ileal submucosal plexus and proximal colon myenteric plexus. At 60 min after injection, CRF decreased VIP levels in the terminal ileum compared with saline (0.8 ± 0.3 vs. 2.5 ± 0.7 ng/g), whereas VIP mRNA level detected by qPCR was not changed. These data indicate that intraperitoneal CRF activates intestinal submucosal VIP neurons most prominently in the ileum and myenteric neurons in the colon. It also implicates VIP signaling as part of underlying mechanisms driving the acute colonic secretomotor response to a peripheral injection of CRF, whereas prostaglandins do not play a role. NEW & NOTEWORTHY Corticotropin-releasing factor (CRF) in the gut plays a physiological role in the stimulation of lower gut secretomotor function induced by stress. We showed that vasoactive intestinal peptide (VIP)-immunoreactive neurons in the ileal submucosal plexus expressed CRF1 receptor and were prominently activated by CRF, unlike colonic submucosal neurons. VIP antagonist abrogated CRF-induced ileal submucosal and colonic myenteric activation along with functional responses (defecation and diarrhea). These data point to VIP signaling in ileum and colon as downstream effectors of CRF.

The antioxidant glutathione protects against enteric neuron death in situ, but its depletion is protective during colitis.

Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor l-buthionine sulfoximine impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Furthermore, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macroscopic and microscopic measures of colonic inflammation. Together, our data highlight the heterogeneous roles of GSH in the myenteric plexus of the ENS and during gastrointestinal inflammation. NEW & NOTEWORTHY Our results show that both enteric glia and neurons express the cellular machinery necessary for glutathione (GSH) synthesis and that glial GSH synthesis is necessary for neuronal survival in isolated enteric nervous system (ENS) preparations. In vivo depletion of GSH with the selective inhibitor l-buthionine sulfoximine is not sufficient to induce inflammation but does alter neuronal neurochemical composition and survival. Together, our data highlight novel heterogeneous roles for GSH in the ENS and during gastrointestinal inflammation.

Physiological Concentrations of Zinc Have Dual Effects on P2X Myenteric Receptors of Guinea Pig.

We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent. The potentiating effect appears to be mediated by allosteric changes induced by Zinc on P2X myenteric channels, which is more frequently observed in myenteric neurons with low zinc concentrations. In P2X2-1 and P2X2-2 variants, the inhibitory effect is more common than in P2X myenteric channels. However, in the variants, the potentiatory effect is of equal magnitude as the inhibitory effect. Inhibitory and potentiatory effects are likely mediated by different binding sites that appear to be present on both P2X2 variants. In conclusion, in myenteric native P2X receptors, Zinc has quantitatively different pharmacological effects compared to those observed on homomeric channels: P2X2-1 and P2X2-2. Potentiatory and inhibitory Zinc effects upon these receptors are mediated by two different binding sites. All our data suggest that myenteric P2X receptors have a more complex pharmacology than those of the recombinant P2X2 receptors, which is likely related to other subunits known to be expressed in myenteric neurons. Because these dual effects occur at Zinc physiological concentrations, we suggest that they could be involved in physiological and pathological processes.

Supplementation with l-glutathione improves oxidative status and reduces protein nitration in myenteric neurons in the jejunum in diabetic Rattus norvegicus.

Diabetes mellitus is a syndrome with multiple etiologies, characterized by chronic hyperglycemia that increases the production of reactive oxygen species and decreases antioxidant defenses. The present study evaluated oxidative stress parameters and protein nitration in myenteric neurons in the jejunum in diabetic rats supplemented with l-glutathione. Rats (90 days of age) were distributed into four groups (n = 6/group): normoglycemic (N), normoglycemic supplemented with l-glutathione (NGT), diabetic (D), and diabetic supplemented with l-glutathione (DGT). At 210 days of age, the animals were sacrificed, and the jejunum was collected, washed, and subjected to various procedures: tert-butyl hydroperoxide chemiluminescence (CL), determination of total antioxidant capacity (TAC), determination of catalase activity, quantification of nitric oxide (NO), and double-labeling of HuC/D-immunoreactive myenteric neurons and nitrotyrosine (3-NT). Diabetes increased oxidative stress in the jejunum in the D group, reflected by increases in lipid peroxidation, TAC, catalase activity, and NO. The D group exhibited an increase in the percentage of myenteric neurons that were double-labeled with 3-NT. Supplementation with l-glutathione did not cause differences in the average CL curves between the D and DGT groups, but reductions of TAC and catalase activity were observed. Supplementation with l-glutathione promoted a reduction of neurons that contained 3-NT in the DGT group. Diabetes mellitus promoted oxidative stress in the jejunum, and supplementation with l-glutathione improved oxidative status by preventing protein nitration in myenteric neurons in diabetic animals that received supplementation.

Effect of CB1 Ligands on Neurogenic and Myogenic Contractile Responses in the Guinea-Pig Ileum.

This study aimed at investigating whether the synthetic cannabinoid receptor agonist (+)-WIN 55212-2 has neurogenic and myogenic relaxant effects on the longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum. (+)-WIN 55212-2, 1-1,000 nmol/L, concentration-dependently inhibited both the electrical stimulation-induced cholinergic twitch responses as well as the myogenic smooth muscle contractions in the LMMP preparation. SR-141716A (rimonabant) 1-1,000 nmol/L, the cannabinoid CB1 receptor antagonist, being without effect on its own, antagonized the (+)-WIN 55212-2-induced effects. The allyl isothiocyanate (mustard oil, 100 µmol/L) induced a relaxant effect in the guinea-pig ileum, which can be regarded as neurogenic and myogenic, was augmented by (+)-WIN 55212-2, and inhibited by SR-141716A. (+)-WIN 55212-2 only moderately modified the 60 mmol/L KCl-evoked contractions. These results provide functional evidence that the CB1 agonist (+)-WIN 55212-2-induced inhibitory effects in the guinea-pig ileum are exerted both at the neuronal as well as at the intestinal smooth muscle cell level.

Self-administration of methamphetamine alters gut biomarkers of toxicity.

Methamphetamine (METH) is a highly abused psychostimulant that is associated with an increased risk for developing Parkinson's disease (PD). This enhanced vulnerability likely relates to the toxic effects of METH that overlap with PD pathology, for example, aberrant functioning of α-synuclein and parkin. In PD, peripheral factors are thought to contribute to central nervous system (CNS) degeneration. For example, α-synuclein levels in the enteric nervous system (ENS) are elevated, and this precedes the onset of motor symptoms. It remains unclear whether neurons of the ENS, particularly catecholaminergic neurons, exhibit signs of METH-induced toxicity as seen in the CNS. The aim of this study was to determine whether self-administered METH altered the levels of α-synuclein, parkin, tyrosine hydroxylase (TH), and dopamine-ß-hydroxylase (DßH) in the myenteric plexus of the distal colon ENS. Young adult male Sprague-Dawley rats self-administered METH for 3 h per day for 14 days and controls were saline-yoked. Distal colon tissue was collected at 1, 14, or 56 days after the last operant session. Levels of α-synuclein were increased, while levels of parkin, TH, and DßH were decreased in the myenteric plexus in the METH-exposed rats at 1 day following the last operant session and returned to the control levels after 14 or 56 days of forced abstinence. The changes were not confined to neurofilament-positive neurons. These results suggest that colon biomarkers may provide early indications of METH-induced neurotoxicity, particularly in young chronic METH users who may be more susceptible to progression to PD later in life.

Abdominal surgery induced gastric ileus and activation of M1-like macrophages in the gastric myenteric plexus: prevention by central vagal activation in rats.

Inflammation plays a role in abdominal surgery (AS)-induced intestinal ileus that is alleviated by electrical vagal stimulation. Intracisternal injection of RX-77368, the stable thyrotropin-releasing hormone agonist, activates dorsal motor nucleus neurons and gastric vagal efferent discharges. We investigated the gastric inflammation induced by AS and the modulation by intracisternal RX-77368 in rats. RX-77368 (50 ng/rat) or saline was injected followed, 1 h later, by laparotomy and small intestinal/cecal manipulation. The sham group had anesthesia alone. After 6 h, gastric emptying (GE) and the inflammation in gastric corpus were determined. AS inhibited GE by 72% vs. control and doubled the number of M1-like macrophage immunoreactive for major histocompatibility complex class II (MHCII; M1 marker) but not for cluster of differentiation 206 (CD206; M2 marker) (MHCII+/CD206-) while there was no change in M2-like macrophages (MHCII-/CD206+). AS increased mRNA levels of interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) by 1.7- and 1.5-fold, respectively, in the gastric submucosa plus muscle layers and the infiltration of neutrophils labeled by myeloperoxidase by 9.5-fold in the muscularis externa. RX-77368 inhibited AS-related gastric changes while not altering these parameters in the sham group. There was a significant negative correlation between GE and IL-1ß (r = -0.46), TNF-α (r = -0.44), M1 macrophage (r = -0.82), and neutrophils (r = -0.91). The M2-like macrophages and IL-10 expression were unchanged by AS with intracisternal saline or RX-77368. These data indicate that AS activates gastric M1 macrophages and increases proinflammatory cytokines expression, which are prevented by central vagal activation and may contribute to the correlated dampening of postoperative gastric ileus.NEW & NOTEWORTHY MHCII+/CD206- (M1) and MHCII-/CD206+ (M2) constitute two distinct populations of macrophages that are in close apposition to the cholinergic neurons in the rat gastric myenteric plexus (MP). Abdominal surgery (6 h) activates M1 macrophage leading to inflammation in the gastric MP correlated with the delayed gastric emptying, which was abolished by central vagal stimulation via intracisternal injection of RX-77368. Vagal stimulation linked with the cephalic phase may have potential beneficial effects to curtail postoperative gastric ileus.

Treatment with low doses of aspirin during chronic phase of experimental Chagas' disease increases oesophageal nitrergic neuronal subpopulation in mice.

Patients with Chagas' disease may develop dysfunctions of oesophageal and colonic motility resulting from the degeneration or loss of the myenteric neurons of the enteric nervous system. Studies have shown that the use of aspirin, also known as acetylsalicylic acid (ASA), influences the pathogenesis of the disease. However, this remains controversial. The aim of this study was to evaluate the consequences of treatment with low doses of aspirin during the chronic phase of Chagas' disease on oesophageal function. Twenty male Swiss mice, 60 days of age, were used. The animals were infected with Y strain of Trypanosoma cruzi, injected intraperitoneally. Aspirin was given at a dose of 50 mg/kg to some of the infected animals, from the 55th to 63rd day after inoculation on consecutive days, and from the 65th to 75th day on alternate days. We investigated food passage of time, wall structure and nitrergic neuronal population of the distal oesophagus. Our data revealed that the use of low doses of aspirin in chronic Chagas' disease caused an increase in the number of nitrergic neurons and partially prevented hypertrophy of the oesophagus. In addition, the aspirin administration impeded Chagas' diseases associated changes in intestinal transit time. Thus treatment with aspirin in the chronic phase of Chagas' disease changes the natural history of the disease and raises the possibility of using it as a new therapeutic approach to the treatment of this aspect of Chagas' disease pathology.

R-Type Ca2+ channels couple to inhibitory neurotransmission to the longitudinal muscle in the guinea-pig ileum.

NEW FINDINGS: What is the central question of this study? Subtypes of enteric neurons are coded by the neurotransmitters they synthesize, but it is not known whether enteric neuron subtypes might also be coded by other proteins, including calcium channel subtypes controlling neurotransmitter release. What is the main finding and its importance? Our data indicate that guinea-pig ileum myenteric neuron subtypes may be coded by calcium channel subtypes. We found that R-type calcium channels are expressed by inhibitory but not excitatory longitudinal muscle motoneurons. R-Type calcium channels are also not expressed by circular muscle inhibitory motoneurons. Calcium channel subtype-selective antagonists could be used to target subtypes of neurons to treat gastrointestinal motility disorders. There is evidence that R-type Ca2+ channels contribute to synaptic transmission in the myenteric plexus. It is unknown whether R-type Ca2+ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase inhibitor nitro-l-arginine (NLA), Ca2+ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea-pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials. Immunohistochemical techniques localized R-type Ca2+ channel protein in the LMMP and circular muscle. Cadmium chloride (pan-Ca2+ channel blocker) blocked and NLA and NiCl2 (R-type Ca2+ channel blocker) reduced neurogenic relaxations in a non-additive manner. Nickel chloride did not alter neurogenic cholinergic contractions, but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl2 and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl2 or ω-conotoxin (N-type Ca2+ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle inhibitory junction potentials were inhibited by NiCl2 but not MRS 2179 (P2Y1 receptor antagonist). Circular muscle inhibitory junction potentials were blocked by apamin, MRS 2179, ω-conotoxin and CdCl2 but not NiCl2 . We conclude that neuronal R-type Ca2+ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea-pig ileum.

Aspirin prevents atrophy of esophageal nitrergic myenteric neurons in a mouse model of chronic Chagas disease.

The consequences of using aspirin (ASA) for the pathogenesis of Chagas disease are unclear. This study evaluated the effects of treatment of Chagas disease with ASA on the esophageal nitrergic myenteric neuron population and esophageal wall in mice. We observed that treatment of chagasic infection with ASA protects the esophageal myenteric neurons from the atrophy caused by the Trypanosoma cruzi infection. The mice were infected with 1300 trypomastigotes of Y strain T. cruzi intraperitoneally. Part of infected mice was treated with ASA from fifth to twelfth day after inoculation. Our data support the hypothesis that eicosanoids given during the acute phase of the chagasic infection may act as immunomodulators aiding the transition to and maintenance of the chronic phase of the disease. Besides, ASA treatment did not provoke alterations in the esophageal wall and the myenteric neurons in infected mice.

A Time-Limited and Partially Reversible Model of Hypoganglionosis Induced by Benzalkonium Chloride Treatment.

Serosal application of benzalkonium chloride (BAC) has been previously applied to produce a model of aganglionosis; however, confusion remains regarding the extent of chemical ablation of enteric myenteric plexus after BAC treatment. The time sequence of BAC-induced effects on the myenteric plexus of the rat colon was determined and followed the morphologic changes. After sacrifice of animals 7, 14, 28, 56, 84 or 168 days postintervention, colonic tissue samples were removed, fixed in formalin, and cut into 5-µm longitudinal sections for histological analysis. The neural analysis was used to re-evaluate BAC treatments for the appropriate model. Compared with rats in sham groups, rats in 0.1 %-30-min BAC group maintained only 15.27 ± 4.80 % of ganglia per section in a 1-cm/5-µm slice and 11.76 ± 2.30 % of ganglionic cells after 28 days, the lower and stable number of ganglionic cells between Day 7 and 84 (from 11.67 ± 2.10 to 19.05 ± 5.10 %). Although an increase, ganglionic cell numbers did not recover at Day168 when compared with the numbers in sham groups. The results showed that characteristics of rats in the 0.1 %-30-min BAC group between Day 7 and 84 most closely kept in stable state, suggesting that these treatment parameters are ideal for producing a hypoganglia model of hypoganglionosis.

Differential effects in CGRPergic, nitrergic, and VIPergic myenteric innervation in diabetic rats supplemented with 2% L-glutamine.

The objective of this study was to investigate the effects of 2% L-glutamine supplementation on myenteric innervation in the ileum of diabetic rats, grouped as follows: normoglycemic (N); normoglycemic supplemented with L-glutamine (NG); diabetic (D); and diabetic supplemented with L-glutamine (DG). The ileums were subjected to immunohistochemical techniques to localize neurons immunoreactive to HuC/D protein (HuC/D-IR) and neuronal nitric oxide synthase enzyme (nNOS-IR) and to analyze varicosities immunoreactive to vasoactive intestinal polypeptide (VIP-IR) and calcitonin gene-related peptide (CGRP-IR). L-Glutamine in the DG group (i) prevented the increase in the cell body area of nNOS-IR neurons, (ii) prevented the increase in the area of VIP-IR varicosities, (iii) did not prevent the loss of HuC/D-IR and nNOS-IR neurons per ganglion, and (iv) reduced the size of CGRP-IR varicosities. L-Glutamine in the NG group reduced (i) the number of HuC/D-IR and nNOS-IR neurons per ganglion, (ii) the cell body area of nNOS-IR neurons, and (iii) the size of VIP-IR and CGRP-IR varicosities. 2% L-glutamine supplementation exerted differential neuroprotective effects in experimental diabetes neuropathy that depended on the type of neurotransmitter analyzed. However, the effects of this dose of L-glutamine on normoglycemic animals suggests there are additional actions of this beyond its antioxidant capacity.