“SPARC” Neurochemical Profile of Enteric Neurons in the Inner and Outer Submucosal Plexus of The Ascending and Descending Colon of Adult Pigs
Authors: Cabanillas Luis , Mazzoni Maurizio , Caremoli Filippo , Ibarra Karla , Lattanzio Giulia , Clavenzani Poalo , De Giorgio Roberto , Sternini Catia 
Published: April 2020

The submucosal plexus of the enteric nervous system plays a major role in the control of mucosal functions. In medium‐size mammals such as the pig, the submucosal plexus is multilayered with an inner submucous plexus (ISP) near the mucosa and an outer submucous plexus (OSP) near the circular muscle, similar to humans. We previously showed that in the adult porcine colon, the number of enteric neurons per mm2 and per ganglion is much higher in the submucosal plexus than in the myenteric plexus (MP), with the highest density in ISP (ISP ≫ OSP >MP). In this study, we compared the neurochemical profile of enteric neurons of the ISP and OSP in the ascending (AC) and descending (DC) colon of the adult pig (male, 25–30 kg Yucatan pigs) using whole mount preparations, multiple labeling immunohistochemistry, high resolution confocal imaging and Imaris software. We used antibodies to HuC/D (as pan‐neuronal marker), choline acetyltransferase (ChAT), neuronal nitric oxide synthase (NOS) and substance P (SP). The ganglia in the ISP are bigger and contain about 3 times the number of cell bodies than those of the OSP with higher density in the AC (ranging from 1100–1200 cells/mm2 in AC vs. 500–600 cells/mm2 in DC) whereas the density of enteric neurons in the OSP was comparable in both AC and DC (ranging 250–400/mm2). The density of cell bodies/ganglion was also higher in the ISP vs. OSP, but there were no differences between AC and DC (129±6 ISP and 37±2 OSP in the AC; 118±11 ISP and 39±4 OSP in the DC). In the ISP, ChAT immunoreactive (IR) neurons were 30–34% with no differences between AC and DC, whereas NOS‐IR neurons were more abundant in DC than AC (42% vs. 15%, p<0.001). In the OSP, ChAT‐ and NOS‐IR neurons were of similar density in AC and DC (~40%). ChAT/NOS‐IR neurons were more abundant in DC vs. AC in ISP (9% vs. 5%), whereas in the OSP, ChAT/NOS‐IR neurons were ~ 10% in both AC and DC, however the differences were not significant. SP‐IR cells were more abundant in the ISP vs. OSP with no significant differences between AC and DC. In the ISP of the AC, SP‐IR was equally localized to a subpopulation of ChAT‐IR, NOS‐IR and ChAT/NOS‐IR neurons, whereas in the DC, SP‐IR was more often observed in ChAT‐IR neurons than in NOS‐IR. Given the location of the two submucosal plexuses, it is reasonable to speculate that the ISP is mostly involved with the regulation of mucosal functions such as absorption, thus explaining the higher abundance of neurons on the ISP of the AC vs. the DC, whereas the OSP contributes to the regulation of both absorption and motility. The characterization of the neurochemical profile of enteric neurons in the pig is important for translational studies on neuronal control of colonic functions in humans since the pig is regarded as a valuable preclinical model for its similarities with humans in physiological and metabolic processes.Support or Funding InformationSupported by NIH SPARC award 1OT2OD24899 (sub‐award to CS) and NIH‐P30DK41301 Imaging Core (CS)