Quantification of nitrergic neurons in the myenteric plexus of gastric antrum and ileum of healthy and diabetic dogs
Graphical abstract
Introduction
The gastrointestinal functions is mainly under the control of the enteric nervous system (ENS), which consists of millions of neurons harbored in the wall of the digestive system from the esophagus to the inner anal sphincter. Enteric neurons are organized in two ganglionated plexuses: the myenteric plexus (MP) and submucosal plexus (SMP), which interact in coordinating gut functions almost independently from the central nervous system (Furness, 2006). The gastrointestinal peristalsis is triggered by sensory fibers responsive to the radial distension of the lumen or by chemical stimuli. Once excited, the intramural sensory neurons activate ENS excitatory and inhibitory muscle motor neurons. The excitatory neurons release acetylcholine, whereas the inhibitory neurons release nitric oxide (Furness, 2006). Nitrergic neurons and fibers, which are usually immunohistochemically identified by the use of an antibody against the enzyme neuronal nitric oxide synthase (nNOS), have been already characterized in the canine gastrointestinal tract (Berezin et al., 1994, Ward et al., 1994). Nevertheless, none of these studies quantified the percentage of nitrergic neurons.
A wide spectrum of damages affecting the structural and functional integrity of the ENS can be responsible for many gastrointestinal symptoms and dysfunction. Among the secondary enteric neuropathies, i.e. heterogeneous disease in which the primary target of the disease is not the ENS (that results however damaged), diabetes mellitus (DM) is classified as a “predominantly degenerative neuropathy” (Knowles et al., 2013).
DM is a worldwide endocrine disease affecting humans but also domestic mammals, such as dogs and cats (Nelson and Reusch, 2014). The common feature of DM is hyperglycemia, which must be controlled to avoid severe DM complications such as retinopathy, vascular damage, generalized neuropathy, and gastrointestinal motility disorders (i.e. vomiting, constipation, diarrhea, and fecal incontinence), in both human and animal models (Rothstein, 1990, Zandecki et al., 2008, Adewoye et al., 2011, Ciobanu and Dumitrascu, 2011). Seemingly, generalized neuropathy and gastrointestinal motility disorders are two strictly correlated complications. As a matter of fact, a growing body of evidence suggests that gastric and intestinal symptoms in human and animal diabetic patients derive from intestinal motility abnormalities related to enteric neuropathy.
A few studies have focused on the gastrointestinal dysfunction in DM dogs (Takeda et al., 2001, Onoma et al., 2008), and to date, no information is available on the effects of DM on canine ENS.
The present research was focused to evaluate whether and how DM affects the dog myenteric neurons and in particular the nitrergic ones, since in other species (mainly rodents) these neurons are susceptible to diabetic neuropathy.
The aims of the present study were to immunohistochemically quantify, in the gastric antrum and ileum of healthy and DM dogs: 1) the density (neurons/ganglionic area) of MP neurons immunoreactive for the pan-neuronal marker HuC/HuD; 2) the percentage of MP nitrergic neurons; 3) the density of nitrergic nervous fibers in the circular (CML) and longitudinal muscle layer (LML).
Section snippets
Animals
Tissues were collected from eight control (CTRL) dogs (none had evident gastrointestinal disorders) (Table 1) and five non-obese DM-affected dogs (Table 2). The weight of DM and CTRL dogs was 18 ± 11 Kg and 25 ± 13 Kg, respectively. Student's t-test did not show any difference between the two groups (P = 0.385). The age of DM and CTRL dogs was 126 ± 63 months and 110 ± 65 months, respectively. Student's t-test did not show any difference between two groups (P = 0.694).
DM type I was diagnosed through a
Pattern of the HuC/HuD immunolabeling
In control dogs, gastric and ileal HuC/HuD immunoreactive (− IR) neurons showed bright and homogenous nuclear and cytoplasmic labeling. In DM dogs, HuC/HuD immunoreactivity was quite preserved in the antrum (Fig. 2 G–I), while in some ileal ganglia, the HuC/HuD-IR was compromised, showing morphological changes. In the worst condition, neuronal somata were barely identifiable and HuC/HuD-labeling seemed dispersed in small granules (Fig. 3 D-L); in some neurons, HuC/HuD-labeling was confined only
Discussion
In the present study, we evaluated, in the antrum and ileum of healthy and diabetic dogs, the density of MP neuronal population, the percentage of nitrergic MP neurons and the density of nitrergic fibers within the muscular layers.
Data obtained in healthy subjects are crucial to compare any alteration during pathological conditions, such as DM.
Our findings indicate that, in either the stomach or ileum of diabetic dogs, although the density of HuC/HuD-IR neurons did not decrease significantly,
Conclusion
The present research turns the spotlight on the effect of spontaneous diabetes on the gastrointestinal intramural innervations, indicating that DM can potentially affect the gastrointestinal motility in dogs.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Conflicts of interest
None.
Author contributions
RC and FF co-designed (conception, planning, and initiation) the study. FF, EZ, GP, and AG recruited animals and defined the clinical features. FG, JS, MA, AG, and GP performed the experiments. RC supervised the experiments. RC and FG analyzed the data and wrote the manuscript. All authors contributed to the interpretation of the data and critically reviewed the manuscript. All authors approved the final version of the manuscript.
Acknowledgments
The excellent technical assistance of Dr. Anna Gardini, Dr. Claudia Vallorani, and Nadia De Sordi is gratefully acknowledged.
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