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Isolation procedure for myenteric plexus from adult mouse after dissection of the gastrointestinal tract: Removal of the mesentery (A), opening of the intestinal segments along the mesenterial line (B), dissecting the muscle from the mucous layer (C) and pulling the muscle sheath without rupturing it (D). The isolated muscle layer was cut into small pieces (E). After the digestion and first mechanical disruption step, the plexus pieces are not yet clearly visible (F, 30x) and have to be further purified (G, 50 x). Propidium iodide (PI) incubation shows only single dead cells in a Nestin-GFP-mouse derived myenteric plexus (H). Magnification 200× scale bar 100 μm. (I–K) Scanning electron microscopical images demonstrate the purity and removal of adjacent cells from the myenteric plexus of the proximal colon. Scale bars (I) 50 μm, (J) 10 μm, (K) 5 μm.
Source publication
The enteric nervous system (ENS) orchestrates a broad range of important gastrointestinal functions such as intestinal motility and gastric secretion. The ENS can be affected by environmental factors, diet and disease. Changes due to these alterations are often hard to evaluate in detail when whole gut samples are used. Analyses based on pure ENS t...
Contexts in source publication
Context 1
... (Nestin-GFP) mice were used in these experiments to dem- onstrate not only the feasibility of the isolation procedure in all seg- ments but also the abundance of intrinsic neural stem cells that can easily be cultured (Fig. 1 ...
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... huge advantage of the described method is the removal of the surrounding cells. Using scanning electron microscopy (SEM), we could clearly demonstrate that smooth muscle cells and connect- ive tissue are completely removed after the digestion procedure ( Fig. 1 SEM pictures). Scanning electron microscopical pictures of the plexus surfaces show only neuronal tissue. ...
Context 3
... are completely removed after the digestion procedure ( Fig. 1 SEM pictures). Scanning electron microscopical pictures of the plexus surfaces show only neuronal tissue. Moreover, western blot analysis of isolated MP did not show smooth muscle actin. The procedure is very gentle and avoids excessive cell death, as demon- strated by live-dead assays (Fig. 1H) and the absence of apoptotic blebbing. The latter could be proven using scanning electron micro- scopy ( Fig. 1 ...
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... procedure is very gentle and avoids excessive cell death, as demon- strated by live-dead assays (Fig. 1H) and the absence of apoptotic blebbing. The latter could be proven using scanning electron micro- scopy ( Fig. 1 I-K). ...
Context 5
... digestion of the tissue with these specific enzymes leads to a much larger amount of isolated MP-networks than classical collage- nase, especially in adult tissues (supplemented Fig. ...
Context 6
... tissue was dehydrated in a graded series of ethanol, starting at 70% for 30 min in each step. The ethanol was then replaced with hexamethyldi- lazane (HMDS) (ethanol/ HMDS1:1 30 min, 2x HMDS 30 min). Finally, the Eppendorf tubes were opened for drying of the MP networks (overnight under a fume hood). ...
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A full-thickness biopsy of the bowel wall is required to evaluate the enteric nervous system. A patient with aggravating gastrointestinal symptoms underwent a laparoscopic full-thickness biopsy of the ileum and, 1 year later, an endoscopic full-thickness biopsy of the sigmoid colon. Both samples showed enteric neuropathy characterized by vacuolated...
Background:
Few studies regarding arthritic diseases have been performed to verify the presence of the neurodegeneration. Given the increased oxidative stress and extra-articular effects of the rheumatoid arthritis, the gastrointestinal studies should be further investigated aiming a better understanding of the systemic effects the disease on ente...
Enteric nervous system (ENS) cells respond to the intestinal extracellular matrix (ECM) signals changing their proliferation rate, migration and differentiation. In this study, we explored in vitro the adaptive response of primary ENS cell cultures to the stimulation of gut acellular matrix (AM) defining the gene expression profile of neuronal func...
The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However,...
Citations
... The tissue samples were created using a whole-mount technique. This technique, although challenging to execute due to the constant rupturing of the myenteric plexus, as already reported by other authors [4,13], allowed much more accurate measurement of the mean area of the neuronal soma. The most common perikaryal size reported in other studies has to be 200 µm 2 to 400 µm 2 [14] that is also confirmed in our study. ...
Ageing-related diseases are paramount for understanding the socioeconomic impact of the postmodern society. Neuronal degeneration is a known phenomenon in ageing. The D-galactose model of accelerated ageing is widely used in age-associated neuronal cell death because of the oxidative stress it induces, which correlates to reduced cognitive performance and robustly detectable neuronal shrinkage in the CNS. This study aims at demonstrating the morphological changes of neurons of the myenteric plexus in mice treated with D-galactose. D-galactose was given orally with drinking water, resulting in an average dose of 500 mg/kg daily for six weeks. In the D-galactose accelerated ageing model, a significant size reduction of the neuronal perikarya of the myenteric plexus was observed all along the large intestine. The average soma area at the caecum in the control group was 305 µm2, which was reduced by almost 20% in the ageing model. In contrast, at the level of the proximal colon in the D-galactose ageing model, the average area of the neuronal soma was 280 µm2, a reduction of 30%. The most significant reduction has been observed at the level of the distal colon, where the neuronal soma was reduced by 40%. There is a significant reduction of the area of the neuronal bodies in the myenteric plexus among different parts of the mice's large intestine in the accelerated ageing model. It can be inferred that D-galactose-induced morphological changes in the myenteric plexus may be related to the increased gastrointestinal motility dysfunction with ageing.
... Myenteric networks were isolated as described previously. 39 Briefly, animals were killed via cervical dislocation and exsanguination, after which the entire colon was removed and transferred to a dish containing ice-cold dissection buffer (Minimal Essential Medium (MEM) with GlutaMAX and HEPES [ThermoFisher 42360032] þ 1% Penicillin and Streptomycin [Pen/Strep]). The mesentery was removed, and the colon was cut open along the mesentery line. ...
Background & Aims
Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system.
Methods
To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways.
Results
Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium–induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia.
Conclusions
Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.
... Last but not least, a greater number of experimental animals is needed contrary to animals used in the 3R principles. As reported by other researchers, during whole-mount preparations, it is not a rare occurrence to rupture the longitudinal muscle layer and even to damage the myenteric plexus [4,5]. We have faced that problem many times in the tissue preparations. ...
... Isolated networks of intact human myenteric ganglia: Isolation of intact human networks of human myenteric plexus ganglia was done according to methods published by Grundmann et al. 51 Networks were loaded with 5µM fluo-4/AM for 45 min and rinsed with Krebs buffer for 45 min for Ca 2+ imaging using the same Nikon Eclipse FN1 microscope imaging system described above. ...
Background and Purpose
ET‐1 signalling modulates intestinal motility and inflammation, but the role of ET‐1/ETB receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ETB signalling regulates neural‐motor pathways of intestinal motility and inflammation.
Experimental Approach
We studied ETB signalling using: ETB drugs (ET‐1, SaTX, BQ788), activity‐dependent stimulation of neurons (high K⁺‐depolarization, EFS), gliotoxins, Tg (Ednrb‐EGFP)EP59Gsat/Mmucd mice, cell‐specific mRNA in Sox10CreERT2;Rpl22‐HAflx or ChATCre;Rpl22‐HAflx mice, Sox10CreERT2::GCaMP5g‐tdT, Wnt1Cre2::GCaMP5g‐tdT mice, muscle tension recordings, fluid‐induced peristalsis, ET‐1 expression, qPCR, western blots, 3‐D LSM‐immunofluorescence co‐labelling studies in LMMP‐CM and a postoperative ileus (POI) model of intestinal inflammation.
Key Results
In the muscularis externa ETB receptor is expressed exclusively in glia. ET‐1 is expressed in RiboTag (ChAT)‐neurons, isolated ganglia and intra‐ganglionic varicose‐nerve fibres co‐labelled with peripherin or SP. ET‐1 release provides activity‐dependent glial ETB receptor modulation of Ca²⁺ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca²⁺ responses and excitatory cholinergic contractions, sensitive to L‐NAME. Gliotoxins disrupt SaTX‐induced glial‐Ca²⁺ waves and prevent BQ788 amplification of contractions. The ETB receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ETB up‐regulation, SaTX‐hypersensitivity and glial amplification of ETB signalling. In vivo BQ788 (i.p., 1 mg·kg⁻¹) attenuates intestinal inflammation in POI.
Conclusion and Implications
Enteric glial ET‐1/ETB signalling provides dual modulation of neural‐motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ETB receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI.
... At the end of the experimental protocol (TNBS: day 7 and 14; EtOH: day 10), mice were sacrificed by decapitation and, from each animal, mesenteric lymph nodes [68], colon [69] and myenteric plexus [70][71][72][73] were collected. In addition, dorsal root ganglia (DRGs) and spinal cord were also isolated from TNBS animals (thoracic-lumbar portion, T10-L1 and lumbar-sacral portion, L6-S1) [74]. ...
Background:
Crohn's disease-(CD) pathogenesis is still unknown and chronic pain is a frequent symptom in CD-patients. Identifying novel therapeutic targets and predisposing factors is a primary goal. In this regard, prokineticin system-(PKS) appears a promising target.
Aims and methods:
TNBS-model was used. DAI, abdominal and visceral pain, and muscle strength were monitored. CD-mice were sacrificed at two times (day 7 and 14 after TNBS) in order to identify PKS involvement in CD pathophysiology and pain. PKS characterization was performed in mesenteric lymph nodes-(MLN), colon, myenteric plexus-(MP), dorsal root ganglia-(DRGs) and spinal cord-(SC). Inflammation/neuroinflammation was also assessed in the same tissues. In order to evaluate alcohol abuse as a possible trigger for CD and its effect on PKS activation, naïve mice were administered (oral-gavage) with ethanol for 10 consecutive days. PKS as well as inflammation/neuroinflammation were evaluated in MLN, colon and MP.
Results:
TNBS treated-mice showed a rapid increase in DAI, abdominal/visceral hypersensitivity and a progressive strength loss. In all tissue analysed of CD-mice, a quick and significant increase of mRNA of PKs and PKRs was observed, associated with an increase of pro-inflammatory cytokines (IL-1β, IL-6 and TNFα) and macrophage/glia markers (iba1, CD11b and GFAP) levels. In alcohol abuse model, ethanol induced in colon and MP a significant PKS activation accompanied by inflammation/neuroinflammation.
Conclusions:
We can assume that PKS may be involved in CD development and pain. Furthermore, alcohol appears to activate PKS and may be a trigger factor for CD.
... Cell Culture Methods: Process described by Grundmann et al. [47] has been used to isolate myenteric plexus from small intestine of postnatal C57BL6J mice (p 2-5). Briefly, muscle layer was stripped and digested for 150 min in Hank's balanced salt solution (H6648, Sigma-Aldrich Corporation), containing collagenase and DNAse. ...
Microelectrode surfaces covered with nanostructures derived from components of extracellular matrix, such as collagen fibers, have shown immense beneficial effects in promoting neuronal growth and cellular signaling. Synthetic nanostructures mimicking the features of biological nanostructures with durable conductive materials could promote the cell adhesion on microelectrode surfaces by providing topographical cues and simultaneously improve the charge transfer properties by reducing its global impedance. Therefore, an advanced nanostructuring method mimicking the structural and organizational features of natural collagen fibers onto metallic microelectrode surfaces has been presented here, which is adapted from previous technological achievements of the group and is based on nanoimprint lithography and gold electroplating. Surface characterization methods reveal an increase in surface area between 20% and 68% on the microelectrodes fabricated with two different nanostructure heights. Impedance spectroscopy measurements reveal reduction in impedance magnitude (at 1 kHz) between 22% and 41%, depending upon the nanostructure height and density on the microelectrode, which should subsequently modulate its charge transfer properties for biosensing application. Cell adhesion analysis performed with seal impedance measurements reveals a tighter coupling of enteric neurons on the nanostructured microelectrodes. Finally, extracellular recordings from enteric neurons exhibit a significant improvement in spike detection properties of the nanostructured microelectrodes.
... While comparative data on digestion methods for ENS isolation from the human gut are lacking, a comprehensive review on the murine intestine recently corroborated the use of Collagenase for this particular purpose (Schonkeren et al, 2021). Importantly, the method described herein is based on full-thickness intestinal biopsies, while the previous work has relied considerably on preparations in which the myenteric plexus was solely dissected and used for subsequent single-cell preparation (Grundmann et al, 2015;Zeisel et al, 2018;Huang et al, 2020;Wright et al, 2021). The latter procedure can result in a biased sampling of EGCs, which are known to be distributed across the entire gut wall axis, but also of enteric neurons present in the submucosa and adjacent to the epithelium (Seguella & Gulbransen, 2021). ...
Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.
... Increase incubation time or enzyme concentration. Liberase allows extended incubation times without risking over digestion (Grundmann et al., 2015). ...
The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated Na V 1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.
... Myentericus plexus cell cultures from p15 Wistar rats were prepared as described before [51,54]. Succinctly, the small intestine from three rats was removed as explained in 4.1, stored in cooled MEM-P/S (MEM-Hepes (M7278, Sigma-Aldrich) containing 1% penicillin-streptomycin (p4333, Sigma-Aldrich) and cut into 3 cm long pieces. ...
Although the enteric nervous system (ENS) functions largely autonomously as part of the peripheral nervous system (PNS), it is connected to the central nervous system (CNS) via the gut–brain axis. In many neurodegenerative diseases, pathological changes occur in addition to gastrointestinal symptoms, such as alpha-synuclein aggregates in Parkinson’s disease, which are found early in the ENS. In both the CNS and PNS, vascular endothelial growth factor (VEGF) mediates neuroprotective and neuroregenerative effects. Since the ENS with its close connection to the microbiome and the immune system is discussed as the origin of neurodegenerative diseases, it is necessary to investigate the possibly positive effects of VEGF on enteric neurons. Using laser microdissection and subsequent quantitative RT-PCR as well as immunohistochemistry, for the first time we were able to detect and localize VEGF receptor expression in rat myenteric neurons of different ages. Furthermore, we demonstrate direct neuroprotective effects of VEGF in the ENS in cell cultures. Thus, our results suggest a promising approach regarding neuroprotection, as the use of VEGF (may) prevent neuronal damage in the ENS.
... Primary enteric cells, in particular MP from newborn and adult mice, were isolated as previously described [26,27]. In brief, stripped muscle layers were cut into small pieces and digested with 0.375 mg/ml Liberase (Roche) and 0.2 mg/ml DNase (Roche) in Hank's balanced salt solution. ...
