DiI-retrograde tracing ex vivo plus immunohistochemistry to characterize longitudinal muscle motor neurons
Study purpose: To characterize longitudinal muscle motor neurons in the human colon.
Data collection: We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter‐tenial longitudinal muscle of human colon.
Primary conclusion: Tenial and inter‐tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized.
Experimental Design: Tissue was obtained from 34 patients undergoing elective surgery with prior written informed consent. The serosa was then dissected to remove connective tissue, and the preparation was washed repeatedly in sterile KREBS solution. For fills from the tenia, a small longitudinal incision was made between bundles of tenial muscle, and the DiI‐coated bead was placed within this slit, allowing contact with axons at multiple levels through the tenia. In order to enhance immunoreactivity for peptides in nerve cell bodies, preparations to be subsequently stained for VIP immunoreactivity were exposed to 83 μmol/L colchicine for the final 24 hours of the culture period. Antibody-stained preparations were viewed on an Olympus IX microscope fit with epifluorescence and appropriate filter sets (Chroma Corporation). Images of labeled neural structures were captured via a CoolSNAP ES digital camera (Roper Scientific, Photometrics) using analySIS 5.0 software (Soft Imaging System). Scanning to identify DiI‐filled nerve cell bodies was carried out with a 10× objective, and cells were photographed with 10‐40× objectives.
Completeness: This dataset is a part of a larger study: "Characterisation of human colon longitudinal muscle motor neurons"
Subjects & Samples: 34 human subjects, 18 female, 16 male, average age 67+/-13 years (mean +/- standard deviation)
Primary vs derivative data: The primary folder consists of original raw data files showing results of tracing enteric neuronal pathways in the human colon, ex vivo, using the carbocyanine dye DiI in combination with dual-labeling immunohistochemistry. Each specific folder includes data from a de-identified patient (sub-) with DiI applied to the circular muscle samples. This was combined with immunohistochemistry for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS). Each sample folder contains also a Microsoft Excel file (.xlsx) with coordinates downloaded from a fluorescence microscope. Visual observations of immunochemical labeled preparations of the human colon from all subjects are summarized in one excel spreadsheet in the pool folder.
Important Notes: This dataset is currently undergoing image registration and will be updated once this is complete.
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