Submucosal neurons and enteric glial cells expressing the P2X7 receptor in rat experimental colitis

Abstract

The aim of this study was to evaluate the effect of ulcerative colitis on the submucosal neurons and glial cells of the submucosal ganglia of rats. 2,4,6-Trinitrobenzene sulfonic acid (TNBS; colitis group) was administered in the colon to induce ulcerative colitis, and distal colons were collected after 24 h. The colitis rats were compared with those in the sham and control groups. Double labelling of the P2X7 receptor with calbindin (marker for intrinsic primary afferent neurons, IPANs, submucosal plexus), calretinin (marker for secretory and vasodilator neurons of the submucosal plexus), HuC/D and S100β was performed in the submucosal plexus. The density (neurons per area) of submucosal neurons positive for the P2X7 receptor, calbindin, calretinin and HuC/D decreased by 21%, 34%, 8.2% and 28%, respectively, in the treated group. In addition, the density of enteric glial cells in the submucosal plexus decreased by 33%. The profile areas of calbindin-immunoreactive neurons decreased by 25%. Histological analysis revealed increased lamina propria and decreased collagen in the colitis group. This study demonstrated that ulcerative colitis affected secretory and vasodilatory neurons, IPANs and enteric glia of the submucosal plexus expressing the P2X7 receptor.

Introduction

Ulcerative colitis and Crohn's disease affect enteric neurons, eventually leading to degeneration of the enteric ganglia (Boyer et al., 2005, da Silva et al., 2015, Geboes and Collins, 1998, Kawada et al., 2007, Linden, 2012, Linden et al., 2005, Lomax et al., 2005b, Sharkey and Kroese, 2001). The enteric nervous system is divided into the submucosal plexus, which controls the transport of fluids to the intestinal mucosa, and the myenteric plexus, which controls intestinal motility (Furness, 2006, Furness, 2012). The submucosal plexus was first described by Meissner (1857) and Billroth (1858) (as cited in Furness, 2006) and is primarily located in the small and large intestines but extends into the esophagus or stomach. The submucosal plexus presents thinner connections than the myenteric plexus, and the associated ganglia are lower than those in the myenteric plexus (Furness, 2006). There are several chemical codes for various neuronal classes in the submucosal plexus; among these are neurons marked by calretinin, which are cholinergic secretomotor and vasodilator neurons that are responsible for the motor and secretory functions of glands and vessels, and neurons marked by calbindin, which function as intrinsic primary afferent neurons (IPANs) (Furness, 2006). Additionally, enteric glial cells can be found in both the submucosal and myenteric ganglia and play a role in neuronal maintenance and the survival of enteric neurons (Gulbransen and Sharkey, 2012, Rühl, 2005).

Adenosine 5′-triphosphate (ATP) receptors, called P2 receptors, are divided into P2X receptors, which are linked to ion channels (ionotropic receptors), and P2Y receptors, which are G-protein coupled (metabotropic receptors) (Burnstock and Kennedy, 1985, Burnstock and Kennedy, 2011). The P2X receptor family includes P2X1-7 (Abbracchio et al., 2009), and the P2Y receptor family includes P2Y1-14 (Abbracchio et al., 2006). P2X receptors have been observed to function in synaptic transmission (Bian et al., 2000, Burnstock, 2014, Galligan, 2002, Galligan et al., 2000).

The P2X7 receptor mediates cell apoptosis, proliferation and inflammation (Burnstock, 2016, Volonté et al., 2012) in addition to neurotransmission (Sperlágh and Illes, 2014, Sperlágh et al., 2006). Neves et al. (2014) have shown that P2X7 receptor levels are increased in the intestinal mucosa of patients with Crohn's disease and in mice with induced colitis, indicating that this receptor may be a therapeutic target. Wan et al. (2016) demonstrated that extracellular ATP levels are increased in experimental colitis mouse models and that the P2X7 receptor antagonist A438079 decreases the activation of NFκB and the expression of caspase-1 in the lamina propria, suggesting that the P2X7 receptor participates in the inflammatory response in experimental colitis. Marques et al. (2014) reported that application of the antagonist A740003 or Brilliant Blue G (BBG) prior to colitis induction exerts a protective effect against inflammation and showed that the levels of NF-kappa-B and Erk in the colon were lower in the colitis animals than in the control animals.

P2X receptors have been documented in the myenteric plexus of a variety of species, including guinea pigs, rats and mice (Castelucci et al., 2002a, Giaroni et al., 2002, Hu et al., 2001, Poole et al., 2002, Ruan and Burnstock, 2005, van Nassauw et al., 2002, Vulchanova et al., 1996, Xiang and Burnstock, 2004a, Xiang and Burnstock, 2004b, Xiang and Burnstock, 2005, Yu et al., 2010).

P2X3 receptor expression is increased in the colon in Crohn’s disease (Yiangou et al., 2001). Alterations of the P2X7 receptor in colon dysfunction have been observed in a rat bowel inflammation model (Antonioli et al., 2014), and the purinergic pathway has been shown to participate in gut diseases (Antonioli et al., 2013). Finally, P2X7 receptor expression has been detected in rat glial cells (Vanderwinden et al., 2003). Additionally, da Silva et al. (2015) observed the effect of colitis in rat myenteric neurons expressing the P2X7 receptor. However, P2X7 receptor expression in the submucosal plexus and in glial cells of the submucosal ganglia in the context of colitis has not been described.

In our work, we studied cholinergic secretomotor and vasodilator submucosal neurons (marked by calretinin), which are responsible for the motor and secretory functions of glands and vessels, and IPANs (marked by calbindin) in the submucosal plexus in the distal colon in a rat ulcerative colitis model. In addition, we characterized the localization of the P2X7 receptor in submucosal neurons and in glial cells of the submucosal ganglia.