Elsevier

Autonomic Neuroscience

Volume 197, May 2016, Pages 25-33
Autonomic Neuroscience

Quantification of nitrergic neurons in the myenteric plexus of gastric antrum and ileum of healthy and diabetic dogs

https://doi.org/10.1016/j.autneu.2016.04.004Get rights and content

Highlights

  • The density of myenteric neurons was not significantly reduced in diabetic (DM) dogs.

  • HuC/HuD immunolabeling showed nuclear dislocation in DM dogs.

  • nNOS-IR myenteric neurons were significantly reduced in the ileum of DM dogs.

  • nNOS-IR nervous fibers were significantly reduced in the muscular layers of DM dogs.

  • Some ileal ganglia of DM dogs showed thickening of the periganglionic connective tissue.

Abstract

Diabetes mellitus (DM) determines a wide array of severe clinical complications including gastrointestinal motility disorders. The present study investigates the effects of spontaneous DM on the intramural innervation and in particular on nitrergic neurons of the myenteric plexus (MP) of the canine gastric antrum and ileum. Specimens of antrum and ileum from eight control-dogs and five insulin-dependent DM-dogs were collected. MP neurons were immunohistochemically identified with the anti-HuC/HuD antibody, while nitrergic neurons were identified with the antibody anti-neuronal nitric oxide synthase (nNOS). The density of HuC/HuD-immunoreactive (IR) neurons was determined and the nitrergic neurons were quantified as a relative percentage, in consideration of the total number of HuC/HuD-IR neurons. Furthermore, the density of nitrergic fibers in the muscular layers was calculated. Data were expressed as mean ± standard deviation. Compared to control-dogs, no significant differences resulted in the density of HuC/HuD-IR neurons in the antrum and ileum of DM-dogs; however, HuC/HuD-immunolabeling showed nuclear localization and fragmentation in DM-dogs. In the stomachs of control- and DM-dogs, the percentages of nitrergic neurons were 30 ± 6% and 25 ± 2%, respectively (P = 0.112). In the ileum of the control-dogs, the percentage of nitrergic neurons was 29 ± 5%, while in the DM-dogs, it was significantly reduced 19 ± 5% (P = 0.006). The density of nNOS-IR nervous fibers was meaningful reduced in either the tracts considered. Notably, the ganglia of DM-dogs showed also a thickening of the periganglionic connective tissue. These findings indicate that DM in dogs induce modification of the myenteric neurons and, in particular, of the nitrergic neuronal subpopulation.

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.

References (42)

  • I. Berezin et al.

    Ultrastructural localization of nitric oxide synthase in canine small intestine and colon

    Am. J. Phys.

    (1994)
  • N. Bodi et al.

    Gut region-specific diabetic damage to the capillary endothelium adjacent to the myenteric plexus

    Microcirculation

    (2012)
  • S.M. Brierley et al.

    Neuroplasticity and dysfunction after gastrointestinal inflammation

    Nat. Rev. Gastroenterol. Hepatol.

    (2014)
  • S. Cellek et al.

    Selective nitrergic neurodegeneration in diabetes mellitus—a nitric oxide-dependent phenomenon

    Br. J. Pharmacol.

    (1999)
  • S. Cellek et al.

    Two phases of nitrergic neuropathy in streptozotocin-induced diabetic rats

    Diabetes

    (2003)
  • S. Cellek et al.

    Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes

    Diabetologia

    (2004)
  • B. Chandrasekharan et al.

    Diabetes and the enteric nervous system

    Neurogastroenterol. Motil.

    (2007)
  • B. Chandrasekharan et al.

    Colonic motor dysfunction in human diabetes is associated with enteric neuronal loss and increased oxidative stress

    Neurogastroenterol. Motil.

    (2011)
  • L. Ciobanu et al.

    Gastrointestinal motility disorders in endocrine diseases

    Pol. Arch. Med. Wewn.

    (2011)
  • I. Demedts et al.

    Gastrointestinal motility changes and myenteric plexus alterations in spontaneously diabetic biobreeding rats

    J. Neurogastroenterol. Motil.

    (2013)
  • A.S. Desmet et al.

    Distinct subcellular localization of the neuronal marker HuC/D reveals hypoxia-induced damage in enteric neurons

    Neurogastroenterol. Motil.

    (2014)
  • Cited by (11)

    • Substance P and the neurokinin-1 receptor expression in dog ileum with and without inflammation

      2017, Research in Veterinary Science
      Citation Excerpt :

      At present, although functional studies on neurokinin receptors exist in dogs (Basilisco and Phillips, 1994; Daniel et al., 1995; Mantyh et al., 1988b; Parlani et al., 1996; Tsukamoto et al., 1997) no morphological findings indicate the subcellular distribution of the other two neurokinin receptors (NK2R and NK3R). During inflammation, the reduction of the percentage of nitrergic neurons bearing the NK1R might be correlated to a general decrease of nNOS-IR neurons, being this neuronal subpopulation susceptible to different pathological conditions (Giancola et al., 2016; Rivera et al., 2011). The presence of NK1R-immunoreactivity on smooth muscle cells is well known in several species (Maggi et al., 1997; Maggi et al., 1990; Portbury et al., 1996; Southwell and Furness, 2001).

    View all citing articles on Scopus
    View full text