scholarly journals Intestinal crypt derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus

2020 ◽  
Author(s):  
Daniel E Levin ◽  
Arabinda Mandal ◽  
Mark A Fleming ◽  
Katherine H Bae ◽  
Brielle Gerry ◽  
...  
Keyword(s):  
Myenteric Plexus ◽  
Longitudinal Muscle ◽  
Culture Media ◽  
Ex Vivo ◽  
Enteric Neurons ◽  
Intestinal Cell ◽  
Cell Populations ◽  
Complex Interactions ◽  
Proliferation And Differentiation ◽  
Intestinal Peristalsis

Abstract The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions is complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: 1) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and 2) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

The Cdx-1 and Cdx-2 homeobox genes in the intestine

1998 ◽  
Vol 76 (6) ◽  
pp. 957-969 ◽  
Author(s):  
Jean-Noël Freund ◽  
Claire Domon-Dell ◽  
Michèle Kedinger ◽  
Isabelle Duluc
Keyword(s):  
Cell Proliferation ◽  
Ex Vivo ◽  
Homeobox Genes ◽  
Intestinal Cell ◽  
Primary Role ◽  
Colon Tumorigenesis ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation

The past years have witnessed an increasing number of reports relative to homeobox genes in endoderm-derived tissues. In this review, we focus on the caudal-related Cdx-1 and Cdx-2 homeobox genes to give an overview of the in vivo, in vitro, and ex vivo approaches that emphasize their primary role in intestinal development and in the control of intestinal cell proliferation, differentiation, and identity. The participation of these genes in colon tumorigenesis and their identification as important actors of the oncogenic process are also discussed.Key words: caudal, epithelial cell proliferation and differentiation, cancer.

scholarly journals Prucalopride exerts neuroprotection in human enteric neurons

2016 ◽  
Vol 310 (10) ◽  
pp. G768-G775 ◽  
Author(s):  
Francesca Bianco ◽  
Elena Bonora ◽  
Dipa Natarajan ◽  
Manuela Vargiolu ◽  
Nikhil Thapar ◽  
...  
Keyword(s):  
Neuronal Degeneration ◽  
Ex Vivo ◽  
Neuroprotective Effect ◽  
Enteric Neurons ◽  
Protective Effects ◽  
Therapeutic Implications ◽  
Sulforhodamine B ◽  
Sulforhodamine B Assay ◽  
Intestinal Peristalsis ◽  
Neuronal Maintenance

Serotonin (5-hydroxytryptamine, 5-HT) and its transporters and receptors are involved in a wide array of digestive functions. In particular, 5-HT4 receptors are known to mediate intestinal peristalsis and recent data in experimental animals have shown their role in neuronal maintenance and neurogenesis. This study has been designed to test whether prucalopride, a well-known full 5-HT4 agonist, exerts protective effects on neurons, including enteric neurons, exposed to oxidative stress challenge. Sulforhodamine B assay was used to determine the survival of SH-SY5Y cells, human enteric neurospheres, and ex vivo submucosal neurons following H2O2 exposure in the presence or absence of prucalopride (1 nM). Specificity of 5-HT4-mediated neuroprotection was established by experiments performed in the presence of GR113808, a 5-HT4 antagonist. Prucalopride exhibited a significant neuroprotective effect. SH-SY5Y cells pretreated with prucalopride were protected from the injury elicited by H2O2 as shown by increased survival (73.5 ± 0.1% of neuronal survival vs. 33.3 ± 0.1%, respectively; P < 0.0001) and a significant reduction of proapoptotic caspase-3 and caspase-9 activation in all neurons tested. The protective effect of prucalopride was reversed by the specific 5-HT4 antagonist GR113808. Prucalopride promotes a significant neuroprotection against oxidative-mediated proapoptotic mechanisms. Our data pave the way for novel therapeutic implications of full 5-HT4 agonists in gut dysmotility characterized by neuronal degeneration, which go beyond the well-known enterokinetic effect.

scholarly journals Influence of Acetylcholine Esterase Inhibitors and Memantine, Clinically Approved for Alzheimer’s Dementia Treatment, on Intestinal Properties of the Mouse

2021 ◽  
Vol 22 (3) ◽  
pp. 1015
Author(s):  
Vu Thu Thuy Nguyen ◽  
Jason Sallbach ◽  
Malena dos Santos Guilherme ◽  
Kristina Endres
Keyword(s):  
Calcium Influx ◽  
Ex Vivo ◽  
Model Organism ◽  
Nmda Receptor Antagonist ◽  
Acetylcholine Esterase ◽  
Enteric Neurons ◽  
Acetylcholine Esterase Inhibitors ◽  
E Coli ◽  
Esterase Inhibitors ◽  
Intestinal Functions

Four drugs are currently approved for the treatment of Alzheimer’s disease (AD) by the FDA. Three of these drugs—donepezil, rivastigmine, and galantamine—belong to the class of acetylcholine esterase inhibitors. Memantine, a NMDA receptor antagonist, represents the fourth and a combination of donepezil and memantine the fifth treatment option. Recently, the gut and its habitants, its microbiome, came into focus of AD research and added another important factor to therapeutic considerations. While the first data provide evidence that AD patients might carry an altered microbiome, the influence of administered drugs on gut properties and commensals have been largely ignored so far. However, the occurrence of digestive side effects with these drugs and the knowledge that cholinergic transmission is crucial for several gut functions enforces the question if, and how, this medication influences the gastrointestinal system and its microbial stocking. Here, we investigated aspects such as microbial viability, colonic propulsion, and properties of enteric neurons, affected by assumed intestinal concentration of the four drugs using the mouse as a model organism. All ex vivo administered drugs revealed no direct effect on fecal bacteria viability and only a high dosage of memantine resulted in reduced biofilm formation of E. coli. Memantine was additionally the only compound that elevated calcium influx in enteric neurons, while all acetylcholine esterase inhibitors significantly reduced esterase activity in colonic tissue specimen and prolonged propulsion time. Both, acetylcholine esterase inhibitors and memantine, had no effect on general viability and neurite outgrowth of enteric neurons. In sum, our findings indicate that all AD symptomatic drugs have the potential to affect distinct intestinal functions and with this—directly or indirectly—microbial commensals.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals Cannabinoid and Cannabinoid-Related Receptors in the Myenteric Plexus of the Porcine Ileum

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 263
Author(s):  
Andrea Toschi ◽  
Giorgia Galiazzo ◽  
Andrea Piva ◽  
Claudio Tagliavia ◽  
Gemma Mazzuoli-Weber ◽  
...  
Keyword(s):  
Myenteric Plexus ◽  
Serotonin Receptor ◽  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Visceral Pain ◽  
Wide Distribution ◽  
Nerve Fibers ◽  
Enteric Neurons ◽  
Beneficial Effects ◽  
Anatomical Basis

An important piece of evidence has shown that molecules acting on cannabinoid receptors influence gastrointestinal motility and induce beneficial effects on gastrointestinal inflammation and visceral pain. The aim of this investigation was to immunohistochemically localize the distribution of canonical cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related receptors transient potential vanilloid receptor 1 (TRPV1), transient potential ankyrin receptor 1 (TRPA1), and serotonin receptor 5-HT1a (5-HT1aR) in the myenteric plexus (MP) of pig ileum. CB1R, TRPV1, TRPA1, and 5-HT1aR were expressed, with different intensities in the cytoplasm of MP neurons. For each receptor, the proportions of the immunoreactive neurons were evaluated using the anti-HuC/HuD antibody. These receptors were also localized on nerve fibers (CB1R, TRPA1), smooth muscle cells of tunica muscularis (CB1R, 5-HT1aR), and endothelial cells of blood vessels (TRPV1, TRPA1, 5-HT1aR). The nerve varicosities were also found to be immunoreactive for both TRPV1 and 5-HT1aR. No immunoreactivity was documented for CB2R. Cannabinoid and cannabinoid-related receptors herein investigated showed a wide distribution in the enteric neurons and nerve fibers of the pig MP. These results could provide an anatomical basis for additional research, supporting the therapeutic use of cannabinoid receptor agonists in relieving motility disorders in porcine enteropathies.

Steerable Needle Trajectory Following in the Lung: Torsional Deadband Compensation and Full Pose Estimation With 5DOF Feedback for Needles Passing Through Flexible Endoscopes

2020 ◽  
Author(s):  
Tayfun Efe Ertop ◽  
Maxwell Emerson ◽  
Margaret Rox ◽  
Josephine Granna ◽  
Robert Webster ◽  
...  
Keyword(s):  
Kalman Filter ◽  
Sliding Mode ◽  
Ex Vivo ◽  
Flexible Bronchoscope ◽  
Complex Interactions ◽  
Trajectory Following ◽  
Steerable Needles ◽  
Needle Trajectory ◽  
Following Error ◽  
Steerable Needle

Abstract Bronchoscopic diagnosis and intervention in the lung is a new frontier for steerable needles, where they have the potential to enable minimally invasive, accurate access to small nodules that cannot be reliably accessed today. However, the curved, flexible bronchoscope requires a much longer needle than prior work has considered, with complex interactions between the needle and bronchoscope channel, introducing new challenges in steerable needle control. In particular, friction between the working channel and needle causes torsional windup along the bronchoscope, the effects of which cannot be directly measured at the tip of thin needles embedded with 5 degree-of-freedom magnetic tracking coils. To compensate for these effects, we propose a new torsional deadband-aware Extended Kalman Filter to estimate the full needle tip pose including the axial angle, which defines its steering direction. We use the Kalman Filter estimates with an established sliding mode controller to steer along desired trajectories in lung tissue. We demonstrate that this simple torsional deadband model is sufficient to account for the complex interactions between the needle and endoscope channel for control purposes. We measure mean final targeting error of 1.36 mm in phantom tissue and 1.84 mm in ex-vivo porcine lung, with mean trajectory following error of 1.28 mm and 1.10 mm, respectively.

TAMI-70. METABOLIC VULNERABILITY TO GPX4 INHIBITION AND FERROPTOSIS OF QUIESCENT ASTROCYTE-LIKE GLIOMA CELL POPULATIONS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi212-vi213
Author(s):  
Matei Banu ◽  
Athanassios Dovas ◽  
Michael Argenziano ◽  
Wenting Zhao ◽  
Dominique Higgins ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Glioma Cell ◽  
Ex Vivo ◽  
Tricarboxylic Acid Cycle ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Acid Oxidation ◽  
Astrocytic Tumor ◽  
Proliferating Cells ◽  
Novel Treatments

Abstract Diversity is a key feature in the glioma ecosystem. Adaptation to a changing tumor microenvironment is achieved through cellular and metabolic plasticity. Here we show that slow-cycling, astrocyte-like glioma cell subpopulations activate distinct metabolic programs, rendering them susceptible to novel treatments. We performed multi-omics analysis on transgenic murine glioma models to characterize cellular heterogeneity. Bulk RNAseq on targeted time-dependent biopsies combined with scRNAseq uncovered distinct tumor cell populations, including a quiescent, astrocyte-like population relatively insensitive to conventional chemotherapy targeting proliferating cells. Using scRNAseq, we identified a persistently conserved astrocytic population in human IDH1-mt/wt high-grade gliomas. This astrocytic tumor population was more abundant in mouse models with constitutive Notch activation, however it was associated with alterations in several other transcriptional programs, suggesting that targeted therapies would likely be ineffective at eradicating it. Gene ontology analysis revealed enrichment in mitochondrial genes specifically regulating oxidative phosphorylation and tricarboxylic acid cycle. Energetic, lipidomic and metabolomic analyses revealed significant mitochondrial β-fatty acid oxidation and lipid catabolism, with less effective oxygen consumption rate and higher basal oxidative stress. Furthermore, this astrocytic tumor population had depleted levels of basal GSH and was more sensitive to reactive oxygen species. Leveraging this metabolic vulnerability, we performed drug screens and found that therapeutic inhibition of complex I or GPX4 was highly effective and synergistic. GPX4 inhibition induced ferroptosis, a newly-discovered form of programmed non-necroptotic cell death mediated by iron-driven lipid peroxidation. Using scRNAseq and RNAscope on ex vivo slice cultures from murine and human gliomas, we found that GPX4 inhibition and ferroptosis induction in the glioma microenvironment selectively eradicated the quiescent astrocytic subpopulation whereas proliferating glioma were less sensitive. Our data therefore supports a novel treatment paradigm, employing metabolic strategies, such as ferroptosis, in conjunction with chemotherapy and RT to target distinct tumor cell populations with different therapeutic vulnerabilities.

Evidence for a glutamatergic neural pathway in the myenteric plexus

1991 ◽  
Vol 261 (4) ◽  
pp. G693-G700 ◽  
Author(s):  
J. W. Wiley ◽  
Y. X. Lu ◽  
C. Owyang
Keyword(s):  
Myenteric Plexus ◽  
Longitudinal Muscle ◽  
Muscle Tension ◽  
Nmda Receptor Antagonist ◽  
Aminobutyric Acid ◽  
Dependent Manner ◽  
Neural Pathway ◽  
Concentration Dependent Manner ◽  
Excitatory Neurotransmitter ◽  
Somatostatin 14

The objective of this study was to determine whether L-glutamate (L-Glu) may serve as a neurotransmitter candidate in the guinea pig myenteric plexus. We observed that [3H]Glu and gamma-[3H]aminobutyric acid were synthesized from [3H]glutamine and released from neurons of the myenteric plexus during K+ and 1,1-dimethyl-4-phenylpiperazinium-evoked depolarization in a concentration-dependent manner. Muscle tension studies performed on ileal longitudinal muscle-myenteric plexus (LM-MP) preparations revealed that L-Glu [mean effective dose (ED50) 2.5 x 10(-5) M] produced concentration-dependent contractions, which were unaffected by hexamethonium but abolished by tetrodotoxin, atropine, and magnesium, suggesting that L-Glu acts via N-methyl-D-aspartate (NMDA)-type receptors that stimulate a cholinergic neural pathway unaffected by ganglionic blockade. In addition, L-Glu (ED50 4 x 10(-5) M) and NMDA (ED50 2 x 10(-4) M) stimulated concentration-dependent release of [3H]acetylcholine (ACh) from LM-MP sections, which was inhibited by tetrodotoxin, magnesium, and the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (AP-5). L-Glu-mediated release of [3H]ACh was enhanced by theophylline (10-6 M) and 3-isobutyl-1-methylxanthine (1 mM) and was significantly reduced by the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (10(-4) M) and somatostatin-14 (10(-6) M), which inhibits adenosine 3',5'-cyclic monophosphate (cAMP)-dependent cholinergic transmission in the myenteric plexus. These studies suggest that L-Glu may serve as an excitatory neurotransmitter in the myenteric plexus via its action on NMDA-type receptors, which are coupled to cAMP-dependent release of ACh.

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