Effects of Chronic Enteropathies on VIPergic and Nitrergic Immunoreactive Neurons in the Dog Ileum

Introduction: The enteric nervous system (ENS) comprises a huge amount of neurons and nerve fibers interposed between the two muscular layers of the tunica muscularis and in the submucosa. Neuropeptides produced by the ENS neurons act as neurotransmitters/neuromodulators, which control intestinal motility and mucosal functions, and play a crucial role also in the regulation of inflammatory processes via cross talk with the local immune system. A growing body of evidence indicates that the gastrointestinal inflammatory response damages the enteric neurons themselves, thus resulting in deregulations in gut motility and mucosal functions. Keywords: Enteric Nervous System; Ileum; Immunohistochemistry; Neuronal Nitric Oxide Synthase; Vasoactive Intestinal Peptide Objective: The purpose of this study was to evaluate quantitatively enteric neurons immunoreactive for the vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the myenteric (MP) and submucosal (SMP) plexus of the ileum of dogs without (CTRL-dogs) and with spontaneous chronic enteritis (inflamed dogs, INF). In addition, the percentage of nNOS immunoreactive neurons co-expressing VIP immunoreactivity (and vice versa) was evaluated. Methods and Material: Animal tissues were collected from the ileum of six control (CTRL) dogs (none had evident gastrointestinal disorders) and ten INF-dogs with chronic enteritis of the ileum. All the enteric neurons, VIPergic and nitrergic neurons were immunohistochemically identified with the anti-HuC/HuD, anti-VIP, and anti-nNOS antibodies, respectively. VIP- and nNOS-immunoreactive neurons were immunohistochemically quantified as a relative percentages, in consideration of the total number of HuC/HuD neurons. Data were expressed as mean ± standard deviation. Results: In the myenteric plexus of INF-dogs, the percentage of VIPergic neurons (16 ± 7%) was significantly greater than that observed in the CTRL-dogs (8 ± 3%) (P = 0,022). Conversely, in the submucosal plexus of CTRL- and INF-dogs the percentages of VIPergic neurons were similar (31 ± 9% and 30 ± 11%, respectively; P = 0,786). In the myenteric plexus of INF-dogs, the percentage of nitrergic neurons (24 ± 5%) showed only a tendency to decrease in comparison to that evaluated in the CTRL-dogs (29 ± 5%) (P = 0.138); also in the submucosal plexus the percentages of nitrergic neurons of CTRL-dogs (8 ± 5%) and INF-dogs (7 ± 2%) did not show meaningful differences (P = 0.884). Co-localization studies indicated that also the percentages of nitrergic neurons co-expressing VIP immunoreactivity did not change between CTRL- and INF-dogs in the MP (23 ± 12% and 24 ± 10%, respectively; P = 0.935) and SMP (26 ± 16% and 23 ± 15%, respectively; P = 0.810). Conclusion: This is the first quantitative study about the VIPergic and nitrergic neurons harbored in the in MP and SMP of the canine ileum and the first comparison between these subclasses of neurons in dogs with and without chronic enteritis. Our findings showed significant