Substance P expression and neurochemical characterization in the enteric nervous system of the porcine colon
Authors: Sternini Catia , Cabanillas Luis , Mazzoni Maurizio , Caremoli Filippo , Million Mulugeta , Larauche Muriel , Clavenzani Paolo , De Giorgio Roberto 
Published: May 2023

The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p<0.01-p<0.0001) with highest density in ISP of AC (p<0.0001 vs. DC). SP-IR neurons represent 27-28% of HuCD-IR neurons in ISP followed by OSP (19-22%) and MP (13-17%, p<0.05- p<0.0001) in AC and DC. The highest proportion of SP-IR neurons contains ChAT-IR (62-67% in the MP, 74-76% in the ISP, 70-85% in OSP) with no significant differences in the AC and DC. By contrast, SP-IR neurons containing nNOS-IR were much less abundant in both AC and DC with the highest peak in the OSP 36% vs. 16% in the ISP, p<0.01, and 20% in the MP in AC, whereas in the DC the highest peak was in the ISP (31%) followed by OSP and MP (25% and 18%) without significant differences. There was a small population of SP-/ChAT-/nNOS-IR enteric neurons in all plexuses in AC and DC. Varicose SP-IR fibers were abundant in the ganglia and distributed to the muscle layers and mucosa. Conclusions: SP-IR neurons include excitatory, inhibitory and interneurons based on the co-expression of ChAT, nNOS or both. The different density and neurochemical profile likely reflect different functions of SP-IR neurons such as immune modulation, regulation of mucosal function and control of vascular, myogenic and neurogenic activities. Mapping neuromodulators expression in a pre-clinical model provides the basis for elucidating neuronal circuits underlying GI functions and advance our understanding of human diseases. Supported by NIH SPARC OT2OD24899 & NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.