Neuroprotective Effect of Quercetin on the Duodenum Enteric Nervous System of Streptozotocin-Induced Diabetic Rats

Background

In diabetes mellitus (DM), hyperglycemia promotes changes in biochemical mechanisms that induce oxidative stress. Oxidative stress has been closely linked to adverse consequences that affect the function of the gastrointestinal tract caused by injuries to the enteric nervous system (ENS) that in turn cause neurodegeneration and enteric glial loss. Therapeutic approaches have shown that diet supplementation with antioxidants, such as quercetin, reduce oxidative stress.

Aims

This work sought to evaluate neurons and enteric glial cells in the myenteric and submucosal plexuses of the duodenum in diabetic rats supplemented with quercetin.

Methods

The duodenum of 24 rats, including a control group (C), control quercetin supplementation group (CQ), diabetic group (D), and diabetic quercetin supplementation group (DQ), were used to investigate whole mounts of muscular and submucosal layers subjected to immunohistochemistry to detect vasoactive intestinal peptide in the myenteric layer and double-staining for HuC-D/neuronal nitric oxide synthase (nNOS) and HuC-D/S100.

Results

A reduction of the general neuronal population (HuC/D) was found in the myenteric and submucosal plexuses (p?<?0.001) in the D and DQ groups. The nitrergic subpopulation (nNOS) decreased only in the myenteric plexus (p?<?0.001), and glial cells decreased in both plexuses (p?<?0.001) in the D and DQ groups. In diabetic rats, quercetin supplementation reduced neuronal and glial loss. Diabetes promoted an increase in the cell body area of both the general and nitrergic populations. Quercetin supplementation only prevented neuronal hypertrophy in the general population.

Conclusion

Supplementation with quercetin eased the damage caused by diabetes, promoting a neuroprotective effect and reducing enteric glial loss in the duodenum.     

Is l-Glutathione More Effective Than l-Glutamine in Preventing Enteric Diabetic Neuropathy?

Background

Diabetes and its complications appear to be multifactorial. Substances with antioxidant potential have been used to protect enteric neurons in experimental diabetes.

Aim

This study evaluated the effects of supplementation with l-glutamine and l-glutathione on enteric neurons in the jejunum in diabetic rats.

Methods

Rats at 90 days of age were distributed into six groups: normoglycemic, normoglycemic supplemented with 2 % l-glutamine, normoglycemic supplemented with 1 % l-glutathione, diabetic (D), diabetic supplemented with 2 % l-glutamine (DG), and diabetic supplemented with 1 % l-glutathione (DGT). After 120 days, the jejunums were immunohistochemically stained for HuC/D+ neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP). Western blot was performed to evaluate nNOS and VIP. Submucosal and myenteric neurons were quantitatively and morphometrically analyzed.

Results

Diabetic neuropathy was observed in myenteric HuC/D, nNOS, and VIP neurons (p < 0.05). In the submucosal plexus, diabetes did not change nitrergic innervation but increased VIPergic neuronal density and body size (p < 0.05). Supplementation with l-glutathione prevented changes in HuC/D neurons in the enteric plexus (p < 0.05), showing that supplementation with l-glutathione was more effective than with l-glutamine. Myenteric nNOS neurons in the DGT group exhibited a reduced density (34.5 %) and reduced area (p < 0.05). Submucosal neurons did not exhibit changes. The increase in VIP-expressing neurons was prevented in the submucosal plexus in the DG and DGT groups (p < 0.05).

Conclusion

Supplementation with l-glutathione exerted a better neuroprotective effect than l-glutamine and may prevent the development of enteric diabetic neuropathy.     

Alterations of the enteric smooth musculature in diverticular disease

Background

The pathogenesis of diverticular disease (DD) is considered to be multifactorial and involves intestinal motor disturbances and an underlying enteric neuromuscular pathology. While an enteric neuropathy has been well documented, actual studies on concomitant alterations of the enteric musculature are limited. This study is aimed at reassessing the smooth muscle tissue by histological, ultrastructural and molecular-biological approaches.

Methods

Full-thickness sigmoid specimens were obtained from patients with DD (n = 20) and controls (n = 19). Morphometric analysis was performed to evaluate the thickness and connective tissue index of the circular and longitudinal muscle layers as well as the myenteric plexus. Structural alterations were determined by light and transmission electron microscopy. mRNA profiles of components of the contractile smooth muscle apparatus including smooth muscle α-actin, smoothelin, histone deacetylase 8, and smooth muscle myosin heavy chain (SMMHC) were assessed by qPCR. Altered gene expression levels were confirmed at protein level by immunohistochemistry.

Results

Compared to controls, patients with DD showed (1) increased thickness of the circular and longitudinal muscle layers, (2) architectural alterations of smooth muscle cells, (3) increased connective tissue index of the longitudinal muscle layer, (4) focally reduced density of myofilaments at ultrastructural level, (5) specific down-regulation of SMMHC mRNA levels, (6) decreased immunoreactivity of SMMHC, (7) oligo-neuronal hypoganglionosis.

Conclusions

DD is associated with distinct structural and functional alterations of the enteric musculature. The enteric myopathy is characterized by disturbed muscular architecture, connective tissue replacement and loss of specific myofilaments and thus may contribute to the pathogenesis and progression of DD.     

Region-specific differences in the human myenteric plexus: an immunohistochemical study using donated elderly cadavers

Purpose and methods

To identify site-dependent and individual differences in neuronal nitric oxide synthase (nNOS)-positive nerves of the myenteric plexus, we examined full-thickness walls of the stomach, pylorus, duodenum, ileum, colon, and rectum in 7 male and 8 female cadavers (mean ages, 80 and 87 years, respectively).

Results

The areas occupied by nNOS-positive nerve fibers in the myenteric plexus were fragmentary and overlapped with areas occupied by vasoactive intestinal polypeptide-positive fibers. The nNOS-positive fiber-containing areas per 1-mm length of intermuscular space tended to be larger at more anal sites, with positive areas four times greater in the rectum than in the stomach. Interindividual differences in rectal areas were extremely large, ranging from 0.017 mm² in one 80-year-old man to 0.067 mm² in another 80-year-old man. Similarly, the numbers of nNOS-positive ganglion cell bodies per 1-mm length in the rectum ranged from 4 to 28. These areas and numbers were weakly correlated (r?=?0.62; p?=?0.02). Interindividual differences in the rectum appeared not to depend on either age or gender.

Conclusions

Anatomic studies using donated cadavers carried the advantage of obtaining any parts of intestine within an individual, in contrast to surgically removed specimens. We speculated excess control of evacuation with laxatives as one of causes of atrophy of the rectal myenteric plexus.     

The microenvironment in the Hirschsprung's disease gut supports myenteric plexus growth

Introduction

The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects.

Methods

Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-β-1 (TGFβ-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated.

Results and conclusions

The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.     

The role of colonic mast cells and myenteric plexitis in patients with diverticular disease

Background

Gut mast cells represent an important cell population involved in intestinal homeostasis and inflammatory processes. However, their possible role has not to date been investigated in colonic diverticular disease.

Aims

This study aims to evaluate colonic mast cells in patients undergoing surgery for diverticular disease.

Methods

Surgical resection samples from 27 patients undergoing surgery for diverticular disease (12 emergency procedures for severe disease and 15 elective procedures) were evaluated. The number of mast cells was assessed in the various layers by means of a specific antibody (tryptase) and compared with those evaluated in ten controls. In patients with mast cells degranulation, double immunohistochemistry, also assessing nerve fibres, was carried out. In addition, the presence of myenteric plexitis was sought.

Results

Compared with controls, the number of mast cells in diverticular patients was significantly increased, both as an overall figure and in the various layers of the large bowel. In patients in whom mast cells degranulation was present, these were always closed to nerve fibres. No differences were found between the two subgroups of patients with respect to the number and distribution of mast cells; however, all patients undergoing emergency surgery (but none of those undergoing elective procedures) had myenteric plexitis, represented by lymphocytic infiltration in 67 % and eosinophilic infiltration in 33 % of cases.

Conclusions

Patients with diverticular disease display an increase of mast cells in the large bowel. The presence of myenteric plexitis in those with complicated, severe disease, suggest that this could represent a histopathologic marker of more aggressive disease.     

Effects of Ischemia and Reperfusion on Subpopulations of Rat Enteric Neurons Expressing the P2X7 Receptor

Background

Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death.

Aim

To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum.

Methods

The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D.

Results

Double immunolabeling demonstrated that 100 % of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6 % in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20 % in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14 % increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14 % increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12 % at 6 h post-I/R and increased by 8 % at 24 h post-I/R.

Conclusions

This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder.     

Exogenous Interleukin-6 Facilitated the Contraction of the Colon in a Depression Rat Model

Background

Gut dysmotility is closely associated with proinflammatory cytokines both in irritable bowel syndrome and inflammatory bowel disease. There is a dose–response relationship between depression and these inflammatory cytokines.

Aims

In the present study, we aimed to investigate the effect of Interleukin-6 (IL-6) on colon motility in a rat model of depression induced by chronic unpredictable mild stress (CUMS).

Methods

The contraction of the circular muscle strips of proximal colon was monitored by a polygraph. IL-6 and IL-6 receptor (IL-6R) mRNA was assayed by real-time quantitative PCR. Immunohistochemistry staining was used to locate the IL-6 and IL-6R in the rat colon.

Results

IL-6 and IL-6R were expressed in the mucosal layer, smooth muscle cells, and myenteric plexus of the colon. Exogenous IL-6 (20 ng/ml) increased the contraction of the circular muscle strip. Pretreatment of tetrodotoxin (blocker of voltage-dependent Na⁺ channel on nerve fiber) blocks the excitatory effect of IL-6 on the contraction of the colon in non-stressed rats, but partially inhibited IL-6-induced excitatory effect on the muscle strips in CUMS-treated rats.

Conclusions

These results suggest that IL-6-induced the contraction of the colonic strip by acting on the gut’s nervous system and acting directly on the smooth muscle in rats with depression.     

Reaggregation of Rat Dissociated Myenteric Plexus in Extracellular Matrix Gels

The aim of this study was to investigate the growth behavior of freshly dissociated myenteric plexus in a three-dimensional extracellular matrix (ECM) environment with and without stimulation of glial cell line-derived neurotrophic factor (GDNF). Therefore, cell suspensions of the dissected myenteric plexus of newborn rats were cultured in freshly prepared gels of commercially available mixtures of collagen, laminin, and hepatoglycans as a first step towards mimicking the natural environment of the myenteric plexus. The cultures were kept either in chemically defined serum-free medium alone or supplemented with GDNF. Cultures on polylysine-coated glass cover slips served as controls. Dissociated myenteric plexus grown on polylysine formed dense clusters of neurons with radially outgrowing nerve fibers, while the neurons cultured in the gel reaggregated to much smaller clusters. These contained, depending on the culture conditions, 2–10 neurons. The morphology of the network that was seen in the gels after a few days in vitro resembled very closely the in situ situation of the submucous plexus and the myenteric plexus in hypoganglionic children. Electron microscope investigations showed a high degree of organization with fiber bundles and vesicle-containing varicosities and growth cones. Independent of the method of culturing, GDNF obviously influenced the growth behavior of the dissociated plexus. The size of the ganglia was larger, and the secondary network denser when GDNF was supplemented. Moreover, the enteric neurons in the gel cultures tended to be larger in size when treated with GDNF. Three-dimensional cultures of dissociated myenteric plexus in an ECM gel might be a valuable tool towards the understanding of the formation of the enteric nervous system during development, especially considering pathological conditions such as Hirschsprungs disease or other dysganglionic diseases.     

Increased Expression of 5-HT3 and NK1 Receptors in 5-Fluorouracil-Induced Mucositis in Mouse Jejunum

Background and Objective

Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80 % of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT₃ and NK₁ receptor expression by immunohistochemical analysis.

Methods

5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay.

Results

The numbers of 5-HT₃ receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT₃ receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK₁ receptor cells in mucosa and immunopositive expression of NK₁ receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT_3A, 5-HT_3B, and NK₁ receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT₃ or NK₁ receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT₃ and NK₁ receptors colocalized with CD11b.

Conclusions

The 5-HT₃ and NK₁ immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.     

Toll-like receptor 4 activation in Barrett’s esophagus results in a strong increase in COX-2 expression

Background

Barrett’s esophagus (BE) is known to progress to esophageal adenocarcinoma in a setting of chronic inflammation. Toll-like receptor (TLR) 4 has been linked to inflammation-associated carcinogenesis. We aimed to determine the expression and functional activity of TLR4 in the esophagus and whether TLR4 activation in BE could promote carcinogenesis by inducing COX-2 expression.

Methods

TLR4 expression in esophageal adenocarcinoma, BE, duodenum, reflux esophagitis and normal squamous esophagus biopsies was assessed using real-time PCR and validated by in situ hybridization and immunohistochemistry. Ex vivo cultures of BE, duodenum and normal squamous esophagus biopsies and a BE cell line (BAR-T) were stimulated with the TLR4 agonist lipopolysaccharide (LPS). To evaluate the effect of TLR4 activation, NF-κB activation, IL8 secretion and expression and COX-2 expression were determined.

Results

TLR4 expression was significantly increased in esophageal adenocarcinoma, BE, duodenum and reflux esophagitis compared to normal squamous esophagus. LPS stimulation resulted in NF-κB activation and a dose-dependent increase of IL8 secretion and mRNA expression. The induction of IL8 was more evident in BE compared to normal squamous esophagus. Upon LPS stimulation, COX-2 expression increased significantly in ex vivo cultured BE biopsies, which was observed in both epithelium and lamina propria cells. However, no effect was found in duodenum and normal squamous esophagus biopsies.

Conclusion

TLR4 activation in BE results in a strong increase in COX-2 and may contribute to malignant transformation.     

Moderate Physical Exercise Protects Myenteric Metabolically More Active Neurons in Mice Infected with Trypanosoma cruzi

Background

Trypanosoma cruzi causes neuronal myenteric depopulation compromising intestinal function.

Aim

The purpose of this study was to evaluate the influence of moderate physical exercise on NADH diaphorase (NADH-d)-positive neurons in the myenteric plexus and intestinal wall of the colon in mice infected with T. cruzi.

Materials and Methods

Forty 30-day-old male Swiss mice were divided into the following groups: trained infected (TI), sedentary infected (SI), trained control (TC), and sedentary control. The TC and TI groups were subjected to a moderate physical exercise program on a treadmill for 8 weeks. Three days after finishing physical exercise, the TI and SI groups were intraperitoneally inoculated with 1,300 blood trypomastigotes of the Y strain of Trypanosoma cruzi. Parasitemia was evaluated from days 4 to 61 after inoculation. On day 75 of infection, myenteric neurons in the colon were quantified (NADH-d), and inflammatory foci were counted. Tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) levels were evaluated in plasma. The results were compared using analysis of variance and the Kruskal–Wallis test at a 5 % significance level.

Results

Moderate physical exercise reduced the parasite peak on day 8 of infection (p = 0.0132) and total parasitemia (p = 0.0307). It also prevented neuronal depopulation (p < 0.01), caused hypertrophy of these cells (p < 0.05), prevented the formation of inflammatory foci (p < 0.01), and increased the synthesis of TNF-α (p < 0.01) and TGF-β (p > 0.05).

Conclusion

These results reinforce the therapeutic benefits of moderate physical exercise for T. cruzi infection.     

Plasticity of Schwann cells and pericytes in response to islet injury in mice

Aims/hypothesis

Islet Schwann (glial) cells and pericytes are the microorgan’s accessory cells positioned at the external and internal boundaries facing the exocrine pancreas and endothelium, respectively, adjacent to the endocrine cells. Plasticity of glial cells and pericytes is shown in the glial scar formation after injury to the central nervous system. It remains unclear whether similar reactive cellular responses occur in insulitis. We applied three-dimensional (3D) histology to perform qualitative and quantitative analyses of the islet Schwann cell network and pericytes in normal, streptozotocin-injected (positive control of gliosis) and NOD mouse models.

Methods

Vessel painting paired with immunostaining of mouse pancreatic tissue was used to reveal the islet Schwann cells and pericytes and their association with vasculature. Transparent islet specimens were prepared by optical clearing to facilitate 3D confocal microscopy for panoramic visualisation of the tissue networks.

Results

In-depth microscopy showed that the islet Schwann cell network extends from the peri-islet domain into the core. One week after streptozotocin injection, we observed intra-islet perivascular gliosis and an increase in pericyte density. In early/moderate insulitis in the NOD mice, perilesional gliosis occurred at the front of the lymphocytic infiltration with atypical islet Schwann cell morphologies, including excessive branching and perivascular gliosis. Meanwhile, pericytes aggregated on the walls of the feeding arteriole at the peri- and intralesional domains with a marked increase in surface marker density.

Conclusions/interpretation

The reactive cellular responses demonstrate plasticity and suggest a stop-gap mechanism consisting of the Schwann cells and pericytes in association with the islet lesion and vasculature when injury occurs.     

In vitro motor patterns and electrophysiological changes in patients with colonic diverticular disease

Purpose

The underlying mechanism responsible for motility changes in colonic diverticular disease (DD) is still unknown. In the present study, our aim was to investigate the structural and in vitro motor changes in the sigmoid colon of patients with DD.

Methods

Muscle bath, microelectrodes and immunohistochemical techniques were performed with samples obtained from the left and sigmoid colon of patients with DD and compared with those of patients without DD.

Results

The amplitude and area under the curve of the spontaneous rhythmic phasic contractions were greatly reduced in patients with DD whereas their frequency and tone remained unaltered. Electrical field stimulation induced a neurally mediated, enhanced ON-contraction (amplitude) in patients with DD and increased the duration of latency of OFF-contractions. The resting membrane potential of smooth muscle cells was hyperpolarized and the amplitude of the inhibitory junction potential was increased in patients with DD. In contrast, no significant histological differences were observed in patients with DD as smooth muscle (circular and longitudinal layers), interstitial cells of Cajal, glial cells and myenteric neurons densities remained unaltered.

Conclusions

Sigmoid strips from patients with asymptomatic DD showed an altered motor pattern with reduced spontaneous motility and enhanced neurally mediated colonic responses involving both excitatory and inhibitory motor pathways. No major neural and muscular structural elements were detected at this stage of the disease. These findings could be valuable in understanding the pathophysiology of this prevalent digestive disease.     

Prevalence and trends of malignant and benign esophageal lesions in Iran between 1997 and 2007

Background

Esophageal cancer is one of the most common malignancies with high mortality worldwide. It comprises two major subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Prevalence of each subtype varies substantially in different geographic regions. In Iran and China, for instance, squamous cell carcinoma predominates. Most Iranian studies regarding benign and malignant lesions of the esophagus to date have focused on the northern part of the country, and a knowledge gap exists regarding the prevalence of esophageal lesions in other parts of the country.

Methods

To assess the prevalence and frequency of benign and malignant esophageal lesions, clinical history of patients who underwent upper gastrointestinal endoscopy and biopsy between 1997 and 2007 in Yazd, Iran was reviewed.

Results

Among 294 malignant lesions, squamous cell carcinoma was the most frequent subtype (70.06?%), followed by adenocarcinoma (29.25?%). Of the 387 benign lesions, most frequent diagnoses were reflux esophagitis 215 (55.5?%), esophagus ulcer 91 (23.51?%), and Barrett??s esophagus 73 (18.86?%). During the 11-year study period, incidence of adenocarcinoma gradually increased over time. On the other hand, squamous cell carcinoma incidence and total incidence of esophageal malignancies did not show a significant trend over time.

Conclusions

Squamous cell carcinoma remains the most common subtype of esophagus malignancies in Yazd, Iran, although an increasing trend towards adenocarcinoma is also evident.     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

Electrophysiological Characteristics of Enteric Neurons Isolated from the Immortomouse

Background

Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2K^b-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line.

Methods

Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 °C and differentiation at 37 °C.

Results

Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was ?24.6 ± 2.1 mV (n = 6) for IM-FEN and ?29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh ?80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh ?80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca²⁺ channels produced large inward currents demonstrating a working mechanism for protein folding and transport.

Conclusion

The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.     

Circuit-specific enteric glia regulate intestinal motor neurocircuits

Glia in the central nervous system exert precise spatial and temporal regulation over neural circuitry on a synapse-specific basis, but it is unclear if peripheral glia share this exquisite capacity to sense and modulate circuit activity. In the enteric nervous system (ENS), glia control gastrointestinal motility through bidirectional communication with surrounding neurons. We combined glial chemogenetics with genetically encoded calcium indicators expressed in enteric neurons and glia to study network-level activity in the intact myenteric plexus of the proximal colon. Stimulation of neural fiber tracts projecting in aboral, oral, and circumferential directions activated distinct populations of enteric glia. The majority of glia responded to both oral and aboral stimulation and circumferential pathways, while smaller subpopulations were activated only by ascending and descending pathways. Cholinergic signaling functionally specifies glia to the descending circuitry, and this network plays an important role in repressing the activity of descending neural pathways, with some degree of cross-inhibition imposed upon the ascending pathway. Glial recruitment by purinergic signaling functions to enhance activity within ascending circuit pathways and constrain activity within descending networks. Pharmacological manipulation of glial purinergic and cholinergic signaling differentially altered neuronal responses in these circuits in a sex-dependent manner. Collectively, our findings establish that the balance between purinergic and cholinergic signaling may differentially control specific circuit activity through selective signaling between networks of enteric neurons and glia. Thus, enteric glia regulate the ENS circuitry in a network-specific manner, providing profound insights into the functional breadth and versatility of peripheral glia.

The nervous system controls essential body functions through neuronal circuits that display exquisite temporal and spatially precise regulation. An emerging body of evidence suggests that glia, which respond to neurotransmitters and release substances that modulate neurotransmission (1, 2), are responsible for at least some of this precision. Glial recruitment by synaptic activity was once considered a general phenomenon that reflects metabolic demands in active neural networks (3, 4). While this may be partially true, more recent data show that signaling in networks of neurons and glia is unexpectedly precise. Astrocytes in particular have emerged as circuit-specific cells that tune neurotransmission on a synapses-by-synapse basis (5–8). These observations provide strong support for the concept that, rather than acting in a purely supportive role, glia serve integral regulatory functions in synaptic circuits. Yet, whether the circuit-specific attributes of astrocytes represent highly evolved functions that emerge in certain circuits in the brain or reflect a more general role of glia in synaptic signaling is not understood.The enteric nervous system (ENS) is the largest and most complex division of the peripheral nervous system. “Brain-like” neurocircuits composed of enteric neurons and glia are intrinsic to the intestine and function to control reflexive activities of the digestive tract such as patterns of intestinal movement (9). The neuronal component of the enteric circuitry is well described, and the characteristics of intrinsic sensory neurons (primary afferent neurons), interneurons, and motor neurons that compose enteric motor circuits are known (10). The basic organization of enteric neurons into functional circuits that control peristalsis is also understood and involves activation of intrinsic primary afferent neurons, which recruit ascending excitatory and descending inhibitory pathways that evoke contractions above and relaxations below the point of stimulation, respectively (10). Although less understood, enteric glia are involved in enteric motor circuits and modulate reflex strength through bidirectional communication with enteric neurons (11–13). During reflexive activities, enteric neurons recruit enteric glia (11, 12), which release transmitters that influence neurotransmission (13, 14). Enteric glia are both necessary (14) and sufficient (13) to control the strength of enteric motility reflexes, but whether subpopulations of enteric glia are devoted to specific synaptic pathways is unknown. Enteric glia are localized to synapses and are postsynaptic to enteric neurons, which places them in an optimal site for regulating the flow of information within the ENS (11, 12, 15). How these interactions ultimately modulate activity within distinct branches of the ENS reflex circuitry remains unclear.The polarized arrangement of enteric synaptic pathways and the ability of isolated preparations to maintain their integrity offers an ideal system for studying the specificity of glial interactions in an integrated yet tractable network. Here, we leveraged this system to test the hypothesis that enteric glia function in a circuit-specific manner. To this end, we evoked activity in overlapping but distinct circuits and recorded calcium (Ca²⁺) responses elicited in the enteric neuron–glial networks involved using genetically encoded Ca²⁺ indicators. We combined this approach with glial chemogenetics to assess whether enteric glia exert specific effects on separate neural pathways and used selective drugs to manipulate cholinergic and purinergic transmitter systems. Our data show that subpopulations of enteric glia are committed to specific ascending or descending neural networks that control intestinal motility. A large subpopulation of glia activated by circumferential pathway stimulation likely represent glial recruitment by intrinsic primary afferent neurons. Furthermore, glial activation exerts differential effects on nearby overlapping functional circuit pathways. The dedication of glia to a particular enteric neurocircuit depends, at least in part, on cholinergic and purinergic transmitter systems that constrain subsets of glia to a particular pathway, effects that are sex dependent. Therefore, bidirectional enteric glia–neuron signaling selectively occurs between specific populations of enteric glia and neurons within distinct functional networks.