Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding

Chen, Bao Nan; Humenick, Adam; Yew, Wai Ping; Peterson, R.A.; Wiklendt, Lukasz; Dinning, Phil G.; Spencer, Nick J.; Wattchow, David; Costa, M.; Brookes, Simon Jonathan

doi:10.1016/j.jcmgh.2023.06.010

Cited by 5 publications

(29 citation statements)

References 82 publications

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“…The solution had been derived from the stripping buffers that are routinely applied to nitrocellulose or polyvinylidene difluoride membranes for re-probing western blots with multiple primary antibodies. The present work and our recent studies [ 4 ],[ 13 , 20 ] showed that this technique also works effectively in wholemount preparations of human colon. In our hands, the method has been tested with up to 9 layers of labelling of wholemount preparations of human gut tissue.…”

Section: Discussionsupporting

confidence: 75%

“…This resistance to elution was exploited in a recent study in which all enteric nerve cell bodies were labelled with the pan-neuronal marker HuCD, visualised with biotinylated secondary and AMCA-coupled streptavidin. The cell bodies could then be individually identified in 5 subsequent layers of labelling in which 12 selective cell body markers were applied, providing a detailed chemical coding profile of each neuron [ 4 ].…”

Section: Discussionmentioning

confidence: 99%

“…In wholemount preparations of enteric plexuses this can be achieved by the application of HuC/D antisera, which label nearly all enteric nerve cell bodies [ 13 , 24 ]. Individual cell bodies can then be reliably re-identified by size, shape and location in ganglia, relative to other cells [ 4 ]. The use of biotinylated secondary antiserum to reveal HuC/D can facilitate this process by making HuC/D labelling resistant to elution so that it persists between layers of antisera without requiring re-staining.…”

Section: Discussionmentioning

confidence: 99%

“…Notwithstanding the power of single cell transcriptome analysis [ 2 ], the “chemical coding” of a neuron (i.e. the combination of neuronal markers that it expresses), has been extensively used as a basis for characterising neurons in both central [ 3 ] and peripheral nervous circuits [ 4 ]. Typically, this is achieved using multiple immunofluorescence labelling to assess co-existence of markers.…”

Section: Introductionmentioning

confidence: 99%

“…Using this modified protocol, 2 layers of antisera were applied to the same preparation, allowing co-localisation of 5 immunohistochemical markers in each myenteric nerve cell body [ 13 ]. In a more recent study, 12 markers were quantified in over 2500 myenteric neurons in wholemount preparations as a basis for dividing them into classes [ 4 ]. Here we report a series of 7 experiments designed to whether 2-ME/SDS works in wholemount preparations of human colonic myenteric plexus, or whether its utility is restricted to paraffin sections examined in previous published studies.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Humenick,

Johnson,

Chen

et al. 2024

Heliyon

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Humenick,

Johnson,

Chen

et al. 2024

Heliyon

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Gut Analysis Toolbox: Automating quantitative analysis of enteric neurons

Sorensen,

Humenick,

Poon

et al. 2024

Preprint

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The enteric nervous system (ENS) plays an important role in coordinating gut function. The ENS consists of an extensive network of neurons and glial cells within the wall of the gastrointestinal tract. Alterations in neuronal distribution, function, and type are strongly associated with enteric neuropathies and gastrointestinal (GI) dysfunction and can serve as biomarkers for disease. However, current methods for assessing neuronal counts and distribution suffer from undersampling. This is partly due to challenges associated with imaging and analyzing large tissue areas, and operator bias due to manual analysis. Here, we present the Gut Analysis Toolbox (GAT), an image analysis tool designed for characterization of enteric neurons and their neurochemical coding using 2D images of GI wholemount preparations. GAT is developed for the Fiji distribution of ImageJ. It has a user-friendly interface and offers rapid and accurate cell segmentation. Custom deep learning (DL) based cell segmentation models were developed using StarDist. GAT also includes a ganglion segmentation model which was developed using deepImageJ. In addition, GAT allows importing of segmentation generated by other software. DL models have been trained using ZeroCostDL4Mic on diverse datasets sourced from different laboratories. This captures the variability associated with differences in animal species, image acquisition parameters, and sample preparation across research groups. We demonstrate the robustness of the cell segmentation DL models by comparing them against the state-of-the-art cell segmentation software, Cellpose. To quantify neuronal distribution GAT applies proximal neighbor-based spatial analysis. We demonstrate how the proximal neighbor analysis can reveal differences in cellular distribution across gut regions using a published dataset. In summary, GAT provides an easy-to-use toolbox to streamline routine image analysis tasks in ENS research. GAT enhances throughput allowing unbiased analysis of larger tissue areas, multiple neuronal markers and numerous samples rapidly.

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From the organ bath to the whole person: a review of human colonic motility

Wattchow,

Brookes,

Spencer

et al. 2023

ANZ Journal of Surgery

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Motor function of the colon is essential for health. Our current understanding of the mechanisms that underlie colonic motility are based upon a range of experimental techniques, including molecular biology, single cell studies, recordings from muscle strips, analysis of part or whole organ ex vivo through to in vivo human recordings. For the surgeon involved in the clinical management of colonic conditions this amounts to a formidable volume of material. Here, we synthesize the key findings from these various experimental approaches so that surgeons can be better armed to deal with the complexities of the colon.

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Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding

Cited by 5 publications

References 82 publications

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Gut Analysis Toolbox: Automating quantitative analysis of enteric neurons

From the organ bath to the whole person: a review of human colonic motility

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