Neurochemical Profile of Enteric Neurons in the Submucosal Plexuses of Pig vs. Human Colon
Authors: Sternini Catia , Cabanillas Luis , Mazzoni Maurizio , Caremoli Filippo , Mulugeta Million , Larauche Muriel , Clavenzani Paolo , De Giorgio Roberto 
Published: May 2022

The enteric nervous system (ENS), the neural network that controls gastrointestinal functions, varies among species. This is particularly evident for the submucosal plexus that is multilayered in humans but single layered and much less developed in rodents and guinea pigs often used for ENS studies. The submucosal plexus plays a major role in the control of gut functions. The pig has many homologies with humans, including a well‐developed submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer submucous plexus (OSP) near the circularmuscle, as in humans. We have previously shown that in the adult pig colon, the density of enteric neurons is significantly higher in the ISP than OSP in ascending (AC) and descending (DC) colon, and that neurons in both plexuses contain choline acetyltransferase (ChAT) immunoreactivity (IR) and neuronal nitric oxide synthase (nNOS)‐IR, markers of the major excitatory (acetylcholine) and inhibitory (nitric oxide) transmitters with a small subpopulation containing both transmitters (Cell Tissue Res. 2021;383(2):645‐654). In this study, we compared the density and neurochemical profile of enteric neurons of the ISP and OSP in the AC and DC of the adult pig (16 Yucatan pigs, 3 F,25‐30 kg) and humans. Human specimens were obtained from 14 patients (6 F, age 48‐86) undergoing elective surgery for colon adenocarcinoma and were taken from the uninvolved resection margin. We used HuC/D (pan‐neuronal marker), ChAT, nNOS and substance P (SP) antibodies in whole mount preparations of the submucosa, confocal imaging and Imaris software for quantification. In both pig and human colon, the highest density of neurons was observed in the ISP of the AC, though the overall density of HuC/D‐IR neurons was about 4 times higher in pig vs. human. As observed in the pig colon, the density of HuC/D‐IR neurons in the ISP of the AC was significantly higher than the ISP of the DC (P<0.01), whereas neuronal density in OSP of AC and DC were not significantly different. The ganglia were also smaller in human ISP and OSP throughout the colon (average ranging from 13‐28 neurons/ganglion) compared to pig where average neurons/ganglion ranged from 30‐120. The % of ChAT‐IR neurons in human (38‐44%) and pig (30‐44%) colon was comparable with no differences between ISP and OSP in AC and DC. The % of nNOS‐IR neurons in human colon was similar in the ISP and OSP of AC and DC (16‐22%), which differs from the pig, where the % of nNOS‐IR neurons in the ISP of AC was significantly lower than OSP (15% vs. 45%) and both ISP and OSP of DC (38‐42%). Overall, the density of nNOS‐IR neurons in human colon was lower compared to the pig colon. The % of SP‐IR neurons was slightly lower in human (15‐20%) vs. pig (23‐26%) colon in both plexuses in the AC and DC. There were dense SP‐IR varicosities through the ganglia in both pig and human colon. SP‐IR mostly colocalized with ChAT‐IR but was also observed in a subpopulation of nNOS‐IR and of nNOS/ChAT‐IR neurons in both human and pig colon. Overall, except for a few differences in neuronal density, there are neurochemical similarities that support the validity of the pig as pre‐clinical model for translational research to explore neuromodulation in colonic disorders in humans.