RESUMEN

Intestinal ischemia-reperfusion (IR) injury is a common gastrointestinal disease that induces severe intestinal dysfunction. Intestinal myenteric neurons participate in maintaining the intestinal function, which will be severely injured by IR. Macrophages are widely reported to be involved in the pathogenesis of organ IR injury, including intestine, which is activated by NLRP3 signaling. Lonicerin (LCR) is a natural extracted monomer with inhibitory efficacy against the NLRP3 pathway in macrophages. The present study aims to explore the potential protective function of LCR in intestinal IR injury. Myenteric neurons were extracted from mice. RAW 264.7 cells were stimulated by H/R with or without 10 µM and 30 µM LCR. Remarkable increased release of IL-6, MCP-1, and TNF-α were observed in H/R treated RAW 264.7 cells, along with an upregulation of NLRP3, cleaved-caspase-1, IL-1ß, and EZH2, which were sharply repressed by LCR. Myenteric neurons were cultured with the supernatant collected from each group. Markedly decreased neuron number and shortened length of neuron axon were observed in the H/R group, which were signally reversed by LCR. RAW 264.7 cells were stimulated by H/R, followed by incubated with 30 µM LCR with or without pcDNA3.1-EZH2. The inhibition of LCR on NLRP3 signaling in H/R treated RAW 264.7 cells was abolished by EZH2 overexpression. Furthermore, the impact of LCR on neuron number and neuron axon length in myenteric neurons in the H/R group was abated by EZH2 overexpression. Collectively, LCR alleviated intestinal myenteric neuron injury induced by H/R treated macrophages via downregulating EZH2.

Asunto(s)

Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2 , Macrófagos , Neuronas , Daño por Reperfusión , Animales , Ratones , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células RAW 264.7 , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Regulación hacia Abajo/efectos de los fármacos , Intestinos/patología , Intestinos/efectos de los fármacos , Plexo Mientérico/metabolismo , Plexo Mientérico/patología , Masculino , Ratones Endogámicos C57BL

RESUMEN

BACKGROUND: Leucine-rich repeat kinase 2 is a molecule that is responsible for familial Parkinson's disease. Our previous findings revealed that leucine-rich repeat kinase 2 is expressed in the enteric nervous system. However, which cells in the enteric nervous system express leucine-rich repeat kinase 2 and whether leucine-rich repeat kinase 2 is associated with the structure of the enteric nervous system remain unclear. The enteric nervous system is remarkable because some patients with Parkinson's disease experience gastrointestinal symptoms before developing motor symptoms. AIMS: We established a leucine-rich repeat kinase 2 reporter mouse model and performed immunostaining in leucine-rich repeat kinase 2 knockout mice. METHODS: Longitudinal muscle containing the myenteric plexus prepared from leucine-rich repeat kinase 2 reporter mice was analyzed by immunostaining using anti-green fluorescent protein (GFP) antibody. Immunostaining using several combinations of antibodies characterizing enteric neurons and glial cells was performed on intestinal preparations from leucine-rich repeat kinase 2 knockout mice. RESULTS: GFP expression in the reporter mice was predominantly in enteric glial cells rather than in enteric neurons. Immunostaining revealed that differences in the structure and proportion of major immunophenotypic cells were not apparent in the knockout mice. Interestingly, the number of biphenotypic cells expressing the neuronal and glial cell markers increased in the leucine-rich repeat kinase 2 knockout mice. Moreover, there was accumulation of α-synuclein in the knockout mice. CONCLUSIONS: Our present findings suggest that leucine-rich repeat kinase 2 is a newly recognized molecule that potentially regulates the integrity of enteric nervous system and enteric α-synuclein accumulation.

Asunto(s)

Sistema Nervioso Entérico , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones Noqueados , Neuronas , alfa-Sinucleína , Animales , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Sistema Nervioso Entérico/metabolismo , Ratones , Neuronas/metabolismo , Plexo Mientérico/metabolismo , Neuroglía/metabolismo , Fenotipo

RESUMEN

The influence of accelerated electrons on neuronal structures is scarcely explored compared to gamma and X-rays. This study aims to investigate the effects of accelerated electron radiation on some pivotal neurotransmitter circuits (cholinergic and serotonergic) of rats' myenteric plexus. Male Wistar rats were irradiated with an electron beam (9 MeV, 5 Gy) generated by a multimodality linear accelerator. The contractile activity of isolated smooth muscle samples from the gastric corpus was measured. Furthermore, an electrical stimulation (200 µs, 20 Hz, 50 s, 60 V) was performed on the samples and an assessment of the cholinergic and serotonergic circuits was made. Five days after irradiation, the recorded mechanical responses were biphasic-contraction/relaxation in controls and contraction/contraction in irradiated samples. The nature of the contractile phase of control samples was cholinergic with serotonin involvement. The relaxation phase involved ACh-induced nitric oxide release from gastric neurons. There was a significant increase in serotonergic involvement during the first and second contractile phases of the irradiated samples, along with a diminished role of acetylcholine in the first phase. This study demonstrates an increased involvement of serotonergic neurotransmitter circuits in the gastric myenteric plexus caused by radiation with accelerated electrons.

Asunto(s)

Electrones , Plexo Mientérico , Ratas Wistar , Estómago , Animales , Plexo Mientérico/efectos de la radiación , Plexo Mientérico/metabolismo , Masculino , Ratas , Estómago/inervación , Estómago/efectos de la radiación , Estómago/fisiología , Músculo Liso/fisiología , Músculo Liso/efectos de la radiación , Músculo Liso/metabolismo , Serotonina/metabolismo , Contracción Muscular/efectos de la radiación , Contracción Muscular/fisiología , Acetilcolina/metabolismo , Óxido Nítrico/metabolismo

RESUMEN

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites ß-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

Asunto(s)

Motilidad Gastrointestinal , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Neuronas , Ratones , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Neuronas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Masculino , Femenino , Animales , Ratones Noqueados , Plexo Mientérico/citología , Plexo Mientérico/metabolismo , Colon/citología , Colon/metabolismo , Membrana Mucosa/citología , Membrana Mucosa/metabolismo

RESUMEN

BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.

Asunto(s)

Adhesión Celular , Técnicas de Cocultivo , Enfermedad de Crohn , Molécula 1 de Adhesión Intercelular , Plexo Mientérico , Neuroglía , Linfocitos T , Adulto , Anciano , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Enfermedad de Crohn/patología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Plexo Mientérico/patología , Plexo Mientérico/metabolismo , Plexo Mientérico/inmunología , Neuroglía/metabolismo , Neuroglía/patología , Neuroglía/inmunología , Estudios Retrospectivos , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission.NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Asunto(s)

Neoplasias Colorrectales , Neuronas Motoras , Ratones , Animales , Inmunohistoquímica , Neurotransmisores/metabolismo , Colinérgicos , Neoplasias Colorrectales/metabolismo , Plexo Mientérico/metabolismo

RESUMEN

BACKGROUND AND AIMS: Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. METHODS: Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. RESULTS: Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. CONCLUSIONS: In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.

Asunto(s)

Colitis Ulcerosa , Colitis , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Plexo Mientérico/metabolismo , Colitis/inducido químicamente , Neurotransmisores/efectos adversos , Dolor , Inflamación

RESUMEN

This study aimed to investigate the distal colon myenteric plexus and enteric glial cells (EGCs) in P2X7 receptor-deficient (P2X7-/-) animals after the induction of experimental ulcerative colitis. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was injected into the distal colon of C57BL/6 (WT) and P2X7 receptor gene-deficient (P2X7-/-, KO) animals. Distal colon tissues in the WT and KO groups were analyzed 24 h and 4 days after administration. The tissues were analyzed by double immunofluorescence of the P2X7 receptor with neuronal nitric oxide synthase (nNOS)-immunoreactive (ir), choline acetyltransferase (ChAT)-ir, and PGP9.5 (pan neuronal)-ir, and their morphology was assessed by histology. The quantitative analysis revealed 13.9% and 7.1% decreases in the number of P2X7 receptor-immunoreactive (ir) per ganglion in the 24 h-WT/colitis and 4 day-WT/colitis groups, respectively. No reduction in the number of nNOS-ir, choline ChAT-ir, and PGP9.5-ir neurons per ganglion was observed in the 4 day-KO/colitis group. In addition, a reduction of 19.3% in the number of GFAP (glial fibrillary acidic protein)-expressing cells per ganglion was found in the 24 h-WT/colitis group, and a 19% increase in the number of these cells was detected in the 4 day-WT/colitis group. No profile area changes in neurons were observed in the 24 h-WT and 24 h-KO groups. The 4 day-WT/colitis and 4 day-KO/colitis groups showed increases in the profile neuronal areas of nNOS, ChAT, and PGP9.5. The histological analysis showed hyperemia, edema, or cellular infiltration in the 24 h-WT/colitis and 4 day-WT/colitis groups. Edema was observed in the 4 day-KO/colitis group, which showed no histological changes compared with the 24 h-KO/colitis group. We concluded that ulcerative colitis differentially affected the neuronal classes in the WT and KO animals, demonstrating the potential participation and neuroprotective effect of the P2X7 receptor in enteric neurons in inflammatory bowel disease.

Asunto(s)

Colitis Ulcerosa , Colitis , Ratones , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Ratones Endogámicos C57BL , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Colitis/metabolismo , Colitis/patología

RESUMEN

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Asunto(s)

Neoplasias Colorrectales , Sistema Nervioso Entérico , Animales , Ratas , Plexo Mientérico/metabolismo , Sistema Nervioso Entérico/metabolismo , Neuronas/metabolismo , Intestinos , Óxido Nítrico Sintasa/metabolismo

RESUMEN

Neurons, expressing neuronal nitric oxide synthase (nNOS) in the enteric ganglia are inhibitory motor neurons or interneurons. The aim of the study was to identify the percentage, cross-sectional area of nNOS-immunoreactive (IR) neurons and their colocalization with choline acetyltransferase (ChAT), vasoactive intestinal polypeptide (VIP), and neuropeptide Y in the intramural ganglia of the myenteric (MP) and submucous plexus (SP) of the small intestine (SI) and large intestine (LI) of rats of different age groups using immunohistochemical methods. In the intramural ganglia of the MP, the largest percentage of nNOS-IR neurons was detected in newborn rats in the LI (81 ± 0.9%) and SI (48 ± 4.1%). Subsequently, it decreased in ontogenesis up to 60 days of life (26 ± 0.9% LI, 29 ± 3.2% SI), and did not change until senescence. In the SP, abundant nNOS-IR neurons were also detected in newborns (82 ± 7.0% SI, 85 ± 3.2% LI), while their percentage decreased significantly in the next 20 days. Furthermore, a very small number of nNOS-IR neurons was detected in 30-day- and 2-month-old animals, but they again appeared in large numbers in aged rats. In the MP, the highest percentage of nNOS+/ChAT+ neurons was in 1-day-old, 10-day-old, and 2-year-old rats. In the SP, the largest number of nNOS-IR neurons colocalized ChAT regardless of age. In the MP of all rats, many nNOS-IR neurons colocalized VIP, and the maximal percentage of nNOS+/VIP+ neurons was found in 2-year-old rats, minimal-in newborns. In conclusion, nNOS expression in neurons of the gut is decreased in early postnatal ontogenesis and subsequently increased in aged rats.

Asunto(s)

Neuronas , Péptido Intestinal Vasoactivo , Animales , Ratas , Óxido Nítrico Sintasa de Tipo I/metabolismo , Neuronas/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Plexo Mientérico/metabolismo , Intestino Delgado/metabolismo

RESUMEN

Huntingtin-associated protein 1(HAP1) is an immunohistochemical marker of the stigmoid body (STB). Brain and spinal cord regions with lack of STB/HAP1 immunoreactivity are always neurodegenerative targets, whereas STB/HAP1 abundant regions are usually spared from neurodegeneration. In addition to the brain and spinal cord, HAP1 is abundantly expressed in the excitatory and inhibitory motor neurons in myenteric plexuses of the enteric nervous system (ENS). However, the detailed expression of HAP1 and its neurochemical characterization in submucosal plexuses of ENS are still unknown. In this study, we aimed to clarify the expression and neurochemical characterization of HAP1 in the submucosal plexuses of the small intestine in adult mice and rats. HAP1 was highly expressed in the submucosal plexuses of both rodents. The percentage of HAP1-immunoreactive submucosal neurons was not significantly varied between the intestinal segments of these rodents. Double immunofluorescence results revealed that almost all the cholinergic secretomotor neurons containing ChAT/ CGRP/ somatostatin/ calretinin, non-cholinergic secretomotor neurons containing VIP/NOS/TH/calretinin, and vasodilator neurons containing VIP/calretinin expressed HAP1. Our current study is the first to clarify that STB/HAP1 is expressed in secretomotor and vasodilator neurons of submucosal plexuses, suggesting that STB/HAP1 might modulate or protect the secretomotor and vasodilator functions of submucosal neurons in ENS.

Asunto(s)

Roedores , Vasodilatadores , Ratas , Ratones , Animales , Calbindina 2/metabolismo , Vasodilatadores/metabolismo , Intestino Delgado , Plexo Mientérico/metabolismo , Neuronas Motoras , Fenotipo

RESUMEN

INTRODUCTION: Calcium-sensitive receptor (CaSR) is expressed in the enteric nervous system of gastrointestinal tract. However, its role in the regulation of gastrointestinal motility has not yet been fully elucidated. We aimed to investigate the effect of the CaSR agonist - R568 on gastric motility and its potential mechanism. METHODS: In vivo, R568 was given by gavage to explore gastric emptying with or without capsaicin which specifically blocks the function of vagal afferents; neurotransmitters synthetized in the myenteric plexus of the gastric corpus and antrum were analysed by ELISA and immunofluorescence staining; gastric muscle strips contraction recording and intracellular single unit firing recording were used to study the effect of R568 on muscle strips and myenteric interstitial cells of Cajal (ICCs) ex vitro. RESULTS: Gastric emptying was inhibited by R568 in Kunming male mice, and capsaicin weakened this effect. The expression of c-fos-positive neurons increased in the nucleus tractus solitarius when R568 was treated. R568 decreased the expression of cholinergic neurons and reduced the synthesis of acetylcholine. Conversely, R568 increased the expression of nitrogenic neurons and enhanced the synthesis of nitric oxide in the myenteric plexus. Ex vitro results showed that R568 inhibited the contraction of the gastric antral muscle strip and suppressed the spontaneous firing activity of pacemaker ICCs. CONCLUSION: Activation of the gastrointestinal CaSR inhibited gastric motility in vivo and ex vitro. Transmitting nutrient signals to the brain through the vagal afferent nerve, modulating the cholinergic and nitrergic system in the enteric nervous system, and inhibiting activity of pacemaker ICCs in the myenteric plexus are involved in the mechanism of CaSR in gastric motility suppression.

Asunto(s)

Calcio , Sistema Nervioso Entérico , Ratones , Animales , Masculino , Calcio/metabolismo , Calcio/farmacología , Capsaicina/farmacología , Capsaicina/metabolismo , Sistema Nervioso Entérico/fisiología , Plexo Mientérico/metabolismo , Motilidad Gastrointestinal/fisiología

RESUMEN

ATP is an excitatory and inhibitory neurotransmitter, while nitric oxide (NO) is an inhibitory neurotransmitter in the enteric nervous system (ENS). We used a vesicular nucleotide transporter (SLC17A9, VNUT) antibody and a nitric oxide synthase (NOS) antibody to identify purinergic and nitrergic nerves in mouse and guinea ileum. Mouse: VNUT-immunoreactivity (ir) was detected in nerve fibers in myenteric ganglia and circular muscle. VNUT-ir fibers surrounded choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and calretinin-ir neurons. VNUT-ir nerve cell bodies were not detected. Tyrosine hydroxylase (TH)-ir nerves were detected in myenteric ganglia and the tertiary plexus. Guinea pig: VNUT-ir was detected in neurons and nerves fibers and did not overlap with NOS-ir nerve fibers. VNUT-ir was detected in nerve fibers in ganglia but not nerve cell bodies. VNUT-ir nerve fibers surrounded NOS-ir and NOS- neurons. NOS-ir and VNUT-ir nerve fibers did not overlap in myenteric ganglia or circular muscle. VNUT-ir nerves surrounded some ChAT-ir neurons. VNUT-ir and ChAT-ir were detected in separate nerves in the CM. VNUT-ir nerve fibers surrounded calretinin-ir neurons.Conclusions: VNUT-ir neurons likely mediate purinergic signaling in small intestinal myenteric ganglia and circular muscle. ATP and NO are likely released from different inhibitory motorneurons. VNUT-ir and ChAT-ir interneurons mediate cholinergic and purinergic synaptic transmission in the myenteric plexus.

Asunto(s)

Plexo Mientérico , Óxido Nítrico Sintasa , Cobayas , Animales , Plexo Mientérico/metabolismo , Calbindina 2 , Óxido Nítrico Sintasa/metabolismo , Músculos/metabolismo , Neurotransmisores , Adenosina Trifosfato

RESUMEN

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

Asunto(s)

Sistema Nervioso Entérico , Plexo Mientérico , Humanos , Ratones , Animales , Plexo Mientérico/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Sistema Nervioso Entérico/metabolismo , Neuronas/fisiología , Colon/metabolismo

RESUMEN

Galanin (GAL) is an important neurotransmitter released by the enteric nervous system (ENS) neurons located in the muscularis externa and submucosa enteric plexuses that acts by binding to GAL receptors 1, 2 and 3 (GALR1, 2 and 3). In our previous studies, the GAL immunoexpression was compared in colorectal cancer (CRC) tissue and the adjacent parts of the large intestine wall including myenteric and submucosal plexuses. Recently we have also found that expression levels of GALR1 and GALR3 proteins are elevated in CRC tissue as compared with their expression in epithelial cells of unchanged mucosa. Moreover, higher GALR3 immunoreactivity in CRC cells correlated with better prognosis of CRC patients. To understand the distribution of GALRs in enteric plexuses distal and close to CRC invasion, in the present study we decided to evaluate GALRs expression within the myenteric and submucosal plexuses located proximally and distally to the cancer invasion and correlated the GALRs expression levels with the clinico-pathological data of CRC patients. The immunohistochemical and immunofluorescent methods showed only slightly decreased immunoexpression of GALR1 and GALR3 in myenteric plexuses close to cancer but did not reveal any correlation in the immunoexpression of all three GAL receptors in myenteric plexuses and tumour progression. No significant changes were found between the expression levels of GALRs in submucosal plexuses distal and close to the tumour. However, elevated GALR1 expression in submucosal plexuses in vicinity of CRC correlated with poor prognosis, higher tumour grading and shorter overall survival. When myenteric plexuses undergo morphological and functional alterations characteristic for atrophy, GALRs maintain or only slightly decrease their expression status. In contrast, the correlation between high expression of GALR1 in the submucosal plexuses and overall survival of CRC patients suggest that GAL and GALRs can act as a components of local neuro-paracrine pro-proliferative pathways accelerating the invasion and metastasis of cancer cell. The obtained results suggest an important role of GALR1 in submucosal plexuses function during the progression of CRC and imply that GALR1 expression in submucosal plexuses of ENS could be an important predictive factor for CRC progression.

Asunto(s)

Neoplasias Colorrectales , Plexo Mientérico , Receptor de Galanina Tipo 1 , Receptor de Galanina Tipo 2 , Receptor de Galanina Tipo 3 , Humanos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Intestinos/inervación , Plexo Mientérico/metabolismo , Receptor de Galanina Tipo 1/metabolismo , Receptor de Galanina Tipo 2/metabolismo , Receptor de Galanina Tipo 3/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia

RESUMEN

Enteric glia are a unique population of peripheral neuroglia that regulate homeostasis in the enteric nervous system (ENS) and intestinal functions. Despite existing in functionally diverse regions of the gastrointestinal tract, enteric glia have been approached scientifically as a homogeneous group of cells. This assumption is at odds with the functional specializations of gastrointestinal organs and recent data suggesting glial heterogeneity in the brain and ENS. Here, we used calcium imaging in transgenic mice of both sexes expressing genetically encoded calcium sensors in enteric glia and conducted contractility studies to investigate functional diversity among myenteric glia in two functionally distinct intestinal organs: the duodenum and the colon. Our data show that myenteric glia exhibit regionally distinct responses to neuromodulators that require intercellular communication with neurons to differing extents in the duodenum and colon. Glia regulate intestinal contractility in a region-specific and pathway-specific manner, which suggests regionally diverse engagement of enteric glia in local motor patterns through discrete signaling pathways. Further, functional response profiles delineate four unique subpopulations among myenteric glia that are differentially distributed between the colon and duodenum. Our findings support the conclusion that myenteric glia exhibit both intraregional and interregional heterogeneity that contributes to region-specific mechanisms that regulate digestive functions. Glial heterogeneity adds an unexpected layer of complexity in peripheral neurocircuits, and understanding the specific functions of specialized glial subtypes will provide new insight into ENS physiology and pathophysiology.SIGNIFICANCE STATEMENT Enteric glia modulate gastrointestinal functions through intercellular communication with enteric neurons. Whether heterogeneity exists among neuron-glia interactions in the digestive tract is not understood. Here, we show that myenteric glia display regional heterogeneity in their responses to neuromodulators in the duodenum and the colon, which are functionally distinct organs. Glial-mediated control of intestinal motility is region and pathway specific. Four myenteric glial subtypes are present within a given gut region that are differently distributed between gut regions. These data provide functional and regional insights into enteric circuit specificity in the adult enteric nervous system.

Asunto(s)

Calcio , Sistema Nervioso Entérico , Masculino , Femenino , Ratones , Animales , Calcio/metabolismo , Neuroglía/metabolismo , Sistema Nervioso Entérico/metabolismo , Colon/fisiología , Duodeno/metabolismo , Neurotransmisores/metabolismo , Ratones Transgénicos , Plexo Mientérico/metabolismo

RESUMEN

CONTEXT.­: Intestinal neuronal dysplasia type B (IND B) is a complex entity involving the enteric nervous system, clinically manifested with constipation in infancy. Diagnosis has been established by histopathologic analysis of rectal biopsies. However, the criteria for the diagnosis have been questioned and modified, hindering diagnostic practice. OBJECTIVE.­: To analyze the applicability of PTEN immunohistochemistry in the diagnosis of IND B and to compare with control cases and cases of Hirschsprung disease (HD). DESIGN.­: PTEN immunohistochemical expression was analyzed in colorectal samples from 29 cases of IND B and compared with 4 control cases and 6 cases of HD. The pattern of PTEN immunoexpression was analyzed in glial cells of the submucosal and myenteric nerve plexuses and in neural fibrils of the muscularis propria using a scoring system. RESULTS.­: Marked reduction or absence of PTEN expression was observed in glial cells of the submucosal nerve plexuses in all cases of the IND B group and in the myenteric nerve plexuses in 28 of 29 cases (96.5%). Lack of PTEN expression was detected in neural fibrils within the muscularis propria in 21 of 29 cases (72%) of the IND B group. PTEN expression was positive in the same neural structures of the control and HD groups. CONCLUSIONS.­: PTEN immunohistochemistry may be a valuable tool in the diagnostic evaluation of IND B. Lack of or reduction of PTEN expression in neural fibrils within the muscularis propria suggests that involvement of the neuromuscular junction may be a key event in the pathogenesis of the motility disturbance occurring in IND B.

Asunto(s)

Sistema Nervioso Entérico , Enfermedad de Hirschsprung , Humanos , Inmunohistoquímica , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/patología , Plexo Mientérico/metabolismo , Plexo Mientérico/patología , Enfermedad de Hirschsprung/complicaciones , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Estreñimiento/patología , Fosfohidrolasa PTEN

RESUMEN

Studied by electron microscopy and morphometry, Auerbach's ganglia comprise nerve cell bodies that occupy ~ 40% of volume; of the neuropil, little over 30% is neural processes (axons, dendrites) and little less than 30% is glia (cell bodies, processes). The amount of surface membrane of neural elements only marginally exceeds that of glia. Glial cells extend laminar processes radially between axons, reaching the ganglion's surface with specialized membrane domains. Nerve cells and glia are tightly associated, eliminating any free space in ganglia. Glia expands maximally its cell membrane with a minimum of cytoplasm, contacting a maximal number of axons, which, with their near-circular profile, have minimal surface for a given volume. Shape of glia is moulded by the neural elements (predominantly concave the first, predominantly convex the second); the glia extends its processes to maximize contact with neural elements. Yet, a majority of axons is not reached by glia and only few are wrapped by it. Despite the large number of cells, the glia is not sufficiently developed to wrap around or just contact many of the neural elements. Mitochondria are markedly fewer in glia than in neurons, indicating a lower metabolic rate. Compactness of ganglia, their near-circular profile, absence of spaces between elements and ability to withstand extensive deformation suggest strong adhesion between the cellular elements, holding them together and keeping them at a fixed distance. Many axonal varicosities, with vesicles and membrane densities, abut on non-specialized areas of glia, suggesting the possibility of neurotransmitters being released outside synaptic sites.

Asunto(s)

Plexo Mientérico , Neuroglía , Animales , Axones/metabolismo , Ganglios/metabolismo , Cobayas , Mitocondrias , Plexo Mientérico/metabolismo , Neuroglía/metabolismo

RESUMEN

The assessment of the enteric nervous system provides a better understanding of the effects that contaminants can have on the health and well-being of organisms. It has been reported that 2,4-dichlorophenoxyacetic acid (2,4-D) is a highly persistent herbicide in the environment that is responsible for neurotoxic changes in different myenteric neuronal subpopulations. The current study aimed to evaluate the effects of 2,4-D on myenteric neurons in the colon of Rattus norvegicus for the first time. A dose of 2,4-D (5 mg/kg/day) was administered to the experimental group (2,4-D) for 15 days. Then, the proximal colon was collected and submitted to Giemsa and NADPH-d histochemical techniques for the disclosure of total and nitrergic neurons. The 2,4-D group presented a higher density of total neurons (p = 0.05, t-test), which together with the maintenance of nitrergic neuronal density, may be related to the increase in the expression of the neurotransmitter acetylcholine by colocalization, responsible for stimulating the intestinal smooth muscle and increasing the chances of the expulsion of the harmful content present in the lumen. Over 15 days, the neurotoxic effects of 2,4-D in the myenteric plexus influenced an increase in the general population of myenteric neurons in the colon.

Asunto(s)

Herbicidas , Neuronas Nitrérgicas , Ácido 2,4-Diclorofenoxiacético/toxicidad , Animales , Colon/metabolismo , Herbicidas/metabolismo , Herbicidas/toxicidad , Humanos , Plexo Mientérico/metabolismo , Neuronas Nitrérgicas/metabolismo , Ratas

RESUMEN

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Asunto(s)

Plexo Mientérico , Óxido Nítrico Sintasa , Animales , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Ratas , Estómago/inervación , Plexo Submucoso