Neurons In Ileum Research Articles

Neuroprotective effects of vitamin D on high fat diet- and palmitic acid-induced enteric neuronal loss in mice

BackgroundThe role of vitamin D in obesity and diabetes is debated. Obese and/or diabetic patients have elevated levels of free fatty acids, increased susceptibility to gastrointestinal symptoms and are suggested to have altered vitamin D balance. The enteric nervous system is pivotal in regulating gastrointestinal activity and high fat diet (HFD) has been shown to cause loss of enteric neurons in ileum and colon. This study investigates the effect of vitamin D on HFD- and palmitic acid-induced enteric neuronal loss in vivo and in vitro.MethodsMice were fed either a normal diet (ND) or HFD supplemented with varying levels of vitamin D (from 0x to 20x normal vitamin D level) for 19 weeks. Ileum and colon were analyzed for neuronal numbers and remodeling. Primary cultures of myenteric neurons from mouse small intestine were treated with palmitic acid (4x10-4M) and/or 1α,25-hydroxy-vitamin D3 (VD, 10-11- 10-7M) with or without modulators of lipid metabolism and VD pathways. Cultures were analyzed by immunocyto- and histochemical methods.ResultsVitamin D supplementation had no effect on enteric neuronal survival in the ND group. HFD caused substantial loss of myenteric neurons in ileum and colon. Vitamin D supplementation between 0-2x normal had no effect on HFD-induced neuronal loss. Supplementation with 20x normal, prevented the HFD-induced neuronal loss. In vitro supplementation of VD prevented the palmitic acid-induced neuronal loss. The VD receptor (VDR) was not identified in enteric neurons. Enteric glia expressed the alternative VD receptor, protein disulphide isomerase family A member 3 (PDIA3), but PDIA3 was not found to mediate the VD response in vitro. Inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) and immune neutralization of isocitrate lyase prevented the VD mediated neuroprotection to palmitic acid exposure.ConclusionsResults show that VD protect enteric neurons against HFD and palmitic acid induced neuronal loss. The mechanism behind is suggested to be through activation of PPARγ leading to improved neuronal peroxisome function and metabolism of neuronal lipid intermediates.

Molecular and pharmacological characterisation of mechano‐gated K‐2P channels in the mouse gastrointestinal tract

Gastrointestinal (GI) motility disorders such as irritable bowel syndrome (IBS) can occur when coordinated smooth muscle contractile activity is disrupted, and little is known about the molecular identity of the potassium (K+) channels that promote GI tract relaxation. Thus, elucidating the expression and function of the potassium channels could reveal novel therapeutic targets to treat spasmodic gut disorders. Here we utilise quantitative RT‐PCR (qPCR), organ bath pharmacology and immunohistochemistry to determine the expression and contribution of K2P channel subtypes in regulating mouse intestinal motility. For isolated organ bath pharmacology, distal ileum and colon segments (2–3cm) were dissected from adult C57BL/6 mice, mounted on an aerator and suspended from a force transducer in an organ bath containing Krebs solution at 32°C. Tension generated from longitudinal muscle contraction was recorded using LabChart® software (AD Instruments). The effects of a variety mechano‐gated K2P channel activators were assessed on sub‐threshold 10 μM carbachol (CCh) or 60 mM KCl pre‐contracted tissue. Data are displayed as mean ± SEM (n = 6, unless stated) and statistical analysis performed by t‐test with significance at p < 0.05. Immunohistochemistry and confocal microscopy was performed on perfusion‐fixed (1% PFA) whole‐mount preparations of the mouse distal ileum and colon. We first performed a qPCR analysis for 14 K2P channel genes on independent ileum and colon sections from 3 animals. KCNK1 (TWIK‐1) and KCNK5 (TASK‐2) showed the greatest relative expression both in ileum and colon, with KCNK2 (TREK‐1) also prominent in ileum but not colon. Given the pause pharmacology for TWIK‐1 and TASK‐2, and previous research highlighting TREK‐1 functionality in colon (1) we pursued the expression pattern and functional role of mechano‐gated K2P channels in gut. 100 μM riluzole caused a significant relaxation in CCh pre‐contracted muscle tension in both ileum (37.1 ± 3.9 %, n = 14, p <0.0001) and colon (18.9 ± 4.4 %, n = 11, p =0.0023). Relaxation induced by 60 μM BL‐1249 and the structurally related fluflenamic acid (100 μM) differed between ileum and colon; 93.1 ± 1.2 % vs 56.1 ± 4.2 % (p <0.001) and 63.3 ± 6.2 % vs 41.1 ± 3.4 % (p = 0.0108) respectively. Immunoreactivity for the TREK‐1 channel was widely distributed across both circular smooth muscle and longitudinal smooth muscle in ileum and colon. Immunoreactivity for the TREK‐2 channel was located on neurons of both the myenteric and submucosal plexuses in ileum and colon, and was associated with NOS, Chat and calretinin‐immunopositive myenteric plexus neurons. TRAAK channel immunoreactivity was evident on NOS, Chat and calretinin‐immunopositive neurons located in both myenteric and submucosal plexus neurons in ileum and colon. These data reveal the molecular and functional diversity of the mechano‐gated K2P channels within the mouse GI tract and highlight the K2P channels as potential therapeutic targets for treating GI motility disorders.

Galectin-3 causes enteric neuronal loss in mice after left sided permanent middle cerebral artery occlusion, a model of stroke.

In addition to brain injury stroke patients often suffer gastrointestinal complications. Neuroimmune interactions involving galectin-3, released from microglia in the brain, mediates the post-stroke pro-inflammatory response. We investigated possible consequences of stroke on the enteric nervous system and the involvement of galectin-3. We show that permanent middle cerebral artery occlusion (pMCAO) induces loss of enteric neurons in ileum and colon in galectin-3+/+, but not in galectin-3−/−, mice. In vitro we show that serum from galectin-3+/+, but not from galectin-3−/−, mice subjected to pMCAO, caused loss of C57BL/6J myenteric neurons, while myenteric neurons derived from TLR4−/− mice were unaffected. Further purified galectin-3 (10−6 M) caused loss of cultured C57BL/6J myenteric neurons. Inhibitors of transforming growth factor β-activated kinase 1 (TAK1) or AMP activated kinase (AMPK) counteracted both the purified galectin-3 and the galectin-3+/+ pMCAO serum-induced loss in vitro. Combined we show that stroke (pMCAO) triggers central and peripheral galectin-3 release causing enteric neuronal loss through a TLR4 mediated mechanism involving TAK1 and AMPK. Galectin-3 is suggested a target for treatment of post-stroke complications.

Effects of Oxaliplatin Treatment on the Enteric Glial Cells and Neurons in the Mouse Ileum.

Oxaliplatin, currently used for treatment of colorectal and other cancers, causes severe gastrointestinal side effects, including nausea, vomiting, diarrhea, and constipation that are attributed to mucosal damage. However, delayed onset and long-term persistence of these side effects suggest that damage to the enteric nervous system (ENS) regulating physiological function of the gastrointestinal tract may also occur. The ENS comprises myenteric and submucosal neurons and enteric glial cells (EGCs). This study aimed to investigate the effects of oxaliplatin treatment on enteric neurons and EGCs within the mouse ileum. BALB/c mice received repeated intraperitoneal injections of oxaliplatin (3 mg/kg, 3 injections/week). Tissues were collected 3, 7, 14, and 21 days from the commencement of treatment. Decreases in glial fibrillary acidic protein-immunoreactive (IR) EGCs and protein gene product 9.5/β-Tubulin III-IR neurons as well as increase in s100β-IR EGCs after chronic oxaliplatin administration were observed in both the myenteric and submucosal plexi. Changes in EGCs were further observed in cross-sections of the ileum at day 14 and confirmed by Western blotting. Alterations in EGCs correlated with loss of myenteric and submucosal neurons in the ileum from oxaliplatin-treated mice. These changes to the ENS may contribute to the mechanisms underlying gastrointestinal side effects associated with oxaliplatin treatment.

Benefits of caloric restriction in the myenteric neuronal plasticity in aging rats.

Aging is a biologic process characterized by progressive damage of structures and functions of organic systems. In gastrointestinal tract, it can involve enteric nervous system, which plays an important role in digestion and absorption of nutrients, causing hastening of intestinal transit thus reducing its absorptive function. Caloric restriction has been used in several studies with the intention of delaying deleterious effects of aging. This study aimed to evaluate the effects of caloric restriction on myenteric neurons of ileum by aging in rats. 30 Wistar rats were grouped as follows: GI (animals aged 6 months fed with normal diet), GII (animals aged 18 months fed with normal diet) and GIII (animals aged 18 months subject to 31% of caloric restriction). The rats of the GI group were euthanized at 6 months of age and after experimental period of 12 months animals of the group GII and GIII were euthanized, the ileum of all groups were collected, measured and processed by NADPH-dp and Acetylcholinesterase. Quantitative analysis of neurons revealed that aging promotes the increasing of myenteric neurons NADPH-dp and reduces Acetylcholinesterase neuronal population. However, in the cellular profile area, were not observed significant differences between the groups. The caloric restriction has been efficient and can be used preventively because it minimizes quantitative changes associated with aging on ileum myenteric plexuses.

Enhanced excitability of guinea pig ileum myenteric AH neurons during and following recovery from chemical colitis

Inflammation of the colon changes motor function of more proximal regions of the gastrointestinal tract. Colitis alters the neurophysiology of enteric neurons within the region of inflammation, which may contribute to altered colonic motor and secretory function. This study seeks to test the hypothesis that colitis alters the neurophysiology of myenteric neurons in the non-inflamed ileum, and that altered neurophysiology coincides with altered small bowel motor function. Trinitrobenzene sulfonic acid (TNBS)-induced colitis was associated with hyperexcitability of AH neurons in the ileum myenteric plexus, demonstrated by depolarized neurons and increased numbers of action potentials, but without changes in the action potential duration or afterhyperpolarization typical of plasticity in these cells. There were no changes in synaptic transmission of either AH neurons or S neurons observed in the current study. The onset of AH neuron hyperexcitability occurred 24h following administration of TNBS, and persisted to eight weeks, a time point following the resolution of colitis. Small bowel transit was reduced as early as 12h after TNBS and resolved by 48h after TNBS. While AH neurons play a central role in coordinating motor function of the ileum, changes in excitability of these neurons did not coincide with changes in small bowel transit.

Ligand-Induced 5-HT3 Receptor Internalization in Enteric Neurons in Rat Ileum

Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs. Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder. Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment. Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.

Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep.

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20-25% of myenteric and 65-75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80+/-13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77+/-7% of CALB-IR cells were NeuN-positive. In mature sheep, 73+/-10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.