Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

Graham, Kahleb; López, Silvia Huerta; Sengupta, Rajarshi; Shenoy, Archana; Schneider, Sabine; Wright, Christina M.; Feldman, Michaël; Furth, Emma E.; Valdivieso, Federico; Lemke, Amanda; Wilkins, Benjamin J.; Naji, Ali; Doolin, Edward J.; Howard, Marthe J.; Heuckeroth, Robert O.

doi:10.1053/j.gastro.2020.02.035

Cited by 48 publications

(63 citation statements)

References 73 publications

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“…Following our new protocol, 13 0.7–1 cm × 0.7–1 cm colon pieces were washed (1× PBS, RT), incubated in 100% methanol on ice (1 hour), treated with Dent's bleach (1 mL 30% hydrogen peroxide (Thermo-Fisher Scientific; Cat. #H1009), 1-mL dimethyl sulfoxide (Sigma-Aldrich; Cat.…”

Section: Methodsmentioning

confidence: 99%

“…We focused on distinctions between myenteric nitrergic (nitric oxide–producing) and cholinergic (acetylcholine-producing) neurons because they comprise >89% of human and >95% of mouse myenteric neuron types. 13 , 14 We found many cell type–specific ion channels, neurotransmitters, adhesion proteins, and signaling pathways supporting functional connectivity, and identified >40 differentially expressed transcriptional regulators, including Tbx3 . Tbx3 is preferentially expressed in neuronal nitric oxide synthase (NOS1)–producing myenteric neurons, and mice lacking Tbx3 in ENS had a 30% reduction in NOS1 neurons.…”

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confidence: 93%

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scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Wright

Schneider

Smith‐Edwards

et al. 2021

Cellular and Molecular Gastroenterology and Hepatology

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113

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Background and Aims Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. Methods To identify subtype–specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. Results RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. Conclusion Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 93%

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Wright

Schneider

Smith‐Edwards

et al. 2021

Cellular and Molecular Gastroenterology and Hepatology

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113

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“…Contrary to the porcine inner submucous plexus, PSI in the human intestine is multi-layered, which means the particular ganglia within this plexus are located at a different depth of the submucous layer [ 56 ]. Other publications have shown that submucosal ganglia in the human colon are disseminated throughout the submucosal layer with significant inter-individual differences [ 60 ].…”

Section: Anatomy Of the Enteric Nervous Systemmentioning

confidence: 99%

Mycotoxins and the Enteric Nervous System

Gonkowski

Gajęcka

Makowska

2020

Toxins

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Mycotoxins are secondary metabolites produced by various fungal species. They are commonly found in a wide range of agricultural products. Mycotoxins contained in food enter living organisms and may have harmful effects on many internal organs and systems. The gastrointestinal tract, which first comes into contact with mycotoxins present in food, is particularly vulnerable to the harmful effects of these toxins. One of the lesser-known aspects of the impact of mycotoxins on the gastrointestinal tract is the influence of these substances on gastrointestinal innervation. Therefore, the present study is the first review of current knowledge concerning the influence of mycotoxins on the enteric nervous system, which plays an important role, not only in almost all regulatory processes within the gastrointestinal tract, but also in adaptive and protective reactions in response to pathological and toxic factors in food.

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“…The volumes of fibroblast-like cells and ganglia were determined, but the whole volume of the ENS in the biopsies was not reported [10,20]. Recently, a different time-consuming method was introduced for confocal microscopic examination after tissue cleaning, immunohistochemistry and quantitative analysis of full thickness human colon, jejunum and pancreas, without embedding and sectioning [21].…”

Section: Discussionmentioning

confidence: 99%

“…At present, double/triple immunohistochemistry, with or without confocal fluorescence microscopy [4,7,8,[25][26][27], is a satisfactory, cheaper and quicker method for this purpose. However, staining and 3D visualization of myenteric ganglia by the new method of Graham and L opez et al [21] takes several weeks, and demands many manual steps. Despite the disadvantages, our long-term goal will be to include more individuals to examine whether 3D X-ray phase-contrast tomography is applicable for the study of gastrointestinal disease mechanisms comparing healthy and diseased tissues.…”

Section: Discussionmentioning

confidence: 99%

3D analysis of the myenteric plexus of the human bowel by X-ray phase-contrast tomography – a future method?

Peruzzi

Veress

Dahlin

et al. 2020

Scandinavian Journal of Gastroenterology

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Objectives: Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. Material and methods: Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton V R tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue ($1mm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 mm 3 (176 nm effective voxel size). Results: The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. Conclusions: Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research.

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Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

Cited by 48 publications

References 73 publications

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

scRNA-Seq Reveals New Enteric Nervous System Roles for GDNF, NRTN, and TBX3

Mycotoxins and the Enteric Nervous System

3D analysis of the myenteric plexus of the human bowel by X-ray phase-contrast tomography – a future method?

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