TITLE: Mechanosensitive enteric neurons: incidence and abundance in the porcine submucosal plexus with ultrafast neuroimaging and immunhistochemical techniques
AUTHORS: Gemma  Mazzuoli-Weber, Kristin Elfers, Anna Katharina Filzmayer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is to investigate the sensitivity of enteric neurons to mechanical stimulation in the inner submucosal plexus of the porcine colon using ultrafast neuroimaging and immunhistochemical techniques. Ultrafast neuroimaging technique combined with a voltage sensitive dye was used to characterize neuronal responses to mechanical stimuli compression and tension. Immunohistochemistry was subsequently used to determine the neurochemical coding of mechanosensitive submucosal neurons.</div></div>

[STEPS]
?. Samples of colon were taken from apparently healthy pigs, placed in ice-cold oxygenated Krebs solution for preparation and immediately transferred to the laboratory. Tissues were then dissected in the ice-cold oxygenated Krebs solution for preparation to obtain whole-mount inner submucosal plexus preparations.
?. Tissue samples were pinned on a ring made of Sylgard®184 and transferred into a recording chamber where they were constantly perfused with 37°C oxygenated experimental Krebs solution.
?. The voltage sensitive dye (VSD) Di-8-ANEPPS was dissolved in 1.5 ml experimental Krebs solution to which 0.135% DMSO and 0.0135% Pluronic® F-127 were added, so that it was used at a concentration of 20 μM.
?. In order to stain selected ganglia, a micropipette was filled with Di-8-ANEPPS and then positioned on the surface of the ganglion. The ganglion sheath was scratched with the tip of the micropipette and the VSD was applied into the ganglion at moderate pressure (≤ 0.5 bar).
?. The viability of ganglia was proven by filling a micropipette with nicotine and then applying nicotine on the surface of a ganglion at a pressure of 0.5 bar for 500 ms.
?. Mechanical stimulation included compression and stretch. Compression of submucosal neurons was induced by filling a micropipette with experimental Krebs solution and then applying it into a ganglion at a pressure of 0.5 bar for 500 ms. Stretch of entire ganglia was performed bidirectionally by moving a self-constructed stretching tool in opposite directions perpendicular to the ganglion axis at a speed of 15 μm/s.
?. The preparations were examined with an inverted microscope equipped with an appropriate filter set. Pictures were acquired with a CMOS camera (DaVinci 1 K) connected to a computer and controlled by Turbo SM 64 Software.
?. Tissue specimens were fixed overnight in a solution containing 4% paraformaldehyde and 0.002% picric acid in 0.1 mol/l phosphate buffer and then washed (3 x 10 min) in PBS.
?. The preparations were then incubated in PBS/NaN3/horse serum for 1h at room temperature followed by 12h and 2h incubation with the primary and secondary antibodies, respectively.
?. Specimens were washed in PBS, mounted on object slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 containing 65% glycerol.
?. The preparations were examined with an epifluorescence microscope equipped with appropriate filter blocks. Pictures were acquired with a camera connected to a computer and controlled by Olympus cellSens Standard Software.
?. For the ganglia examined the following applied: based on their localisation in the fixed tissue preparation and their specific morphology, they could be clearly identified.
?. Mechanosensitive neurons previously determined by ultrafast neuroimaging technique and subsequently stained with the antibodies could be clearly identified based on their localization in the fixed tissue preparation and their specific morphology. Pictures of the stained ganglia were acquired.