Elsevier

Neuroscience

Volume 290, 2 April 2015, Pages 357-368
Neuroscience

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression

https://doi.org/10.1016/j.neuroscience.2015.01.056Get rights and content

Highlights

  • Regeneration of axons restores damage from intestinal inflammation.

  • A mechanism related to proliferation of intestinal smooth muscle is proposed.

  • Smooth muscle proliferation in vitro and in vivo causes GDNF expression.

  • GDNF expression supports enteric neurons and axon outgrowth in co-culture.

  • Phenotype modulation causes failure of GDNF expression and loss of neural support.

Abstract

Intestinal inflammation causes initial axonal degeneration and neuronal death, as well as the proliferation of intestinal smooth muscle cells (ISMC), but subsequent axonal outgrowth leads to re-innervation. We recently showed that expression of glial cell-derived neurotrophic factor (GDNF), the critical neurotrophin for the post-natal enteric nervous system (ENS) is upregulated in ISMC by inflammatory cytokines, leading us to explore the relationship between ISMC growth and GDNF expression. In co-cultures of myenteric neurons and ISMC, GDNF or fetal calf serum (FCS) was equally effective in supporting neuronal survival, with neurons forming extensive axonal networks among the ISMC. However, only GDNF was effective in low-density cultures where neurons lacked contact with ISMC. In early-passage cultures of colonic circular smooth muscle cells (CSMC), polymerase chain reaction (PCR) and western blotting showed that proliferation was associated with expression of GDNF, and the successful survival of neonatal neurons co-cultured on CSMC was blocked by vandetanib or siGDNF. In tri-nitrobenzene sulfonic acid (TNBS)-induced colitis, immunocytochemistry showed the selective expression of GDNF in proliferating CSMC, suggesting that smooth muscle proliferation supports the ENS in vivo as well as in vitro. However, high-passage CSMC expressed significantly less GDNF and failed to support neuronal survival, while expressing reduced amounts of smooth muscle marker proteins. We conclude that in the inflamed intestine, smooth muscle proliferation supports the ENS, and thus its own re-innervation, by expression of GDNF. In chronic inflammation, a compromised smooth muscle phenotype may lead to progressive neural damage. Intestinal stricture formation in human disease, such as inflammatory bowel disease (IBD), may be an endpoint of failure of this homeostatic mechanism.

Introduction

Inflammatory diseases of the gastrointestinal tract commonly affect the mucosa, but also challenge the normal homeostatic balance of all parts of the intestine, including the neurons of the enteric nervous system (ENS), such as those in the myenteric plexus and their target of innervations – the intestinal smooth muscle cells (ISMC) in the muscularis externa. Smooth muscle hypertrophy and hyperplasia as well as altered neuronal structure are described in the inflamed intestine in both human inflammatory bowel disease (IBD) and in animal disease models. In the well-studied tri-nitrobenzene sulfonic acid (TNBS)-induced model of colitis, there is a significant loss of myenteric neurons at the onset of intestinal inflammation in the rat, mouse and guinea pig. For example, we showed a loss of 50% of these neurons within two days following the induction of colitis in rats, with no significant loss thereafter, despite ongoing and worsening inflammation over the subsequent 4–6 days (Sanovic et al., 1999). Subsequently, Linden et al. (2005) showed indiscriminate loss of neurons in the guinea pig model that was maintained by 56 days post colitis, and Boyer et al. (2005) showed the involvement of activated neutrophils in mediating damage to myenteric neurons in a mouse model. Recently, we showed that the initial rapid influx of activated immune cells was responsible for the loss of myenteric neurons in both mouse and rat models of TNBS-induced colitis, and used inhibitors of inducible nitric oxide synthase to selectively prevent this loss (Venkataramana et al., 2015).

The loss of enteric neurons causes a sharp decrease in axonal projections to smooth muscle targets, resulting in denervation of this tissue at early stages. Subsequently, axon growth by surviving myenteric neurons into the surrounding tissues returns the innervation density to control levels by Day 6 of colitis (Lourenssen et al., 2005). While recent evidence has identified immunological factors contributing to the loss of ENS neurons (see above), there are major unanswered questions about the mechanisms influencing the remaining neurons, which limit the initial loss and sustain their subsequent survival as well as promote axonal outgrowth.

Growth factors and neurotrophic factors have been demonstrated to be responsible for the development of the neurons and precursor cells in the embryonic intestine, such as endothelin-3, NT-3 and glial cell-derived neurotrophic factor (GDNF), among others (Lake and Heuckeroth, 2013). In these studies, gene-deletion mouse mutants have shown that GDNF, its GPI-anchored binding receptor, GFRα-1 and its transducing receptor tyrosine kinase, Ret, are essential for normal ENS development. Further, a recent study has shown that the hindgut is colonized by a GDNF-dependent migration of neural crest cells across the mesentery from the midgut to the hindgut, and in addition, a conditional GFRα-1 knockout showed that this receptor was essential for the transmigration process (Nishiyama et al., 2012). Previously, our laboratory showed that GDNF is a major neurotrophin in the postnatal gut that supports survival of postnatal enteric neurons in vitro (Rodrigues et al., 2011). In this study, GDNF was expressed by postnatal ISMC both in vitro and in vivo.

While ISMC proliferate during post-natal development to create the adult intestine, these cells are quiescent in the adult animal, with little or no detectable proliferation (Blennerhassett et al., 1992). However, inflammation causes the rapid onset of ISMC dedifferentiation and proliferation (e.g., by Day 2 of TNBS-colitis (Lourenssen et al., 2005, Stanzel et al., 2010, Nair et al., 2011)), which leads to a large and essentially permanent increase in ISMC number. Nonetheless, simultaneous proliferation of axons exactly matches this, achieving an outcome of innervation density that is identical to the start-point (Lourenssen et al., 2005). The mechanisms involved in this tight regulation are unknown but are likely to involve signaling between ENS neurons and ISMC via GDNF.

The major factor influencing the onset of proliferation by ISMC is acquisition of sensitivity to the known smooth muscle mitogen PDGF-BB, via the onset of expression of its receptor, PDGF-Rβ (Stanzel et al., 2010, Nair et al., 2014). This receptor is normally not expressed in these cells, but appears among control cells shortly after enzymatic isolation and tissue culture, as well as by 24 h after intracolonic TNBS administration in the rat. Further, the normal innervation of ISMC also maintains these cells in a quiescent, differentiated state and disruption of innervation promotes hyperplasia and phenotypic modulation (Blennerhassett and Lourenssen, 2000, Pelletier et al., 2010).

As well as increased number, ISMC proliferation during inflammation has consequences that remain after the resolution of inflammation. This includes reduced expression of smooth muscle markers such as α-smooth muscle actin, desmin and SM22, as well as a decreased cholinergic sensitivity (Nair et al., 2011). Overall, better understanding of intrinsic mechanisms that promote neuronal survival and repair could have broad effects on amelioration of the damaging and lasting effects of an inflammatory episode.

Since proliferating ISMC have the potential for GDNF expression during growth in the postnatal intestine, we proposed that adult, quiescent ISMC would again express this neurotrophin when caused to proliferate, as occurs during inflammation in vivo. This might be part of the alteration of gene expression that is associated with the transition to the proliferative state, and is normally reversed with cessation of growth (Alexander and Owens, 2012). However, extended proliferation of smooth muscle cells can cause further modulation of phenotype that leads to irreversible changes, which are observed both in vitro and in disease states in vivo. Therefore, we also tested the consequences of repeated passaging of cultured adult ISMC on GDNF expression. For these hypotheses, we used an established primary co-culture model of isolated enteric neurons, smooth muscle cells and glia cells as well as ISMC isolated from the adult circular smooth muscle layer of the adult rat. The TNBS-induced model of colitis in the rat was used to validate these findings in vivo, with the overall goal of better understanding of the outcomes of denervation and altered ISMC phenotype that are present in chronic intestinal stricturing disease (Marlow and Blennerhassett, 2006).

Section snippets

Animals

Adult BalbC mice (20–25 g) and Sprague–Dawley rats (200–225 g) were obtained from Charles River (Quebec, Canada) and bred in our animal facility. Colitis was induced in male mice by instillation of 100 μL of 5% (w/v) TNBS (Sigma, St. Louis, MO, USA) dissolved in 50% ethanol into the colon 4 cm proximal to the anus. Colons were removed at Day 2 post-TNBS, and processed for routine embedding in paraffin.

Animals were sacrificed by cervical dislocation under isoflurane anesthesia. All procedures

Results

The neurotrophin GDNF was shown earlier to support the survival and axonal outgrowth of neonatal myenteric neurons in vitro, while other neurotrophins were ineffective (Rodrigues et al., 2011). In the co-culture model of neonatal myenteric neurons, smooth muscle and glia, GDNF clearly improved the outcome of neurons compared with culture medium alone (DMEM), with immunocytochemistry showing that neuron number was increased and that axon number and length was much more substantial (Fig. 1 A, B).

Discussion

The embryonic development of the ENS depends on expression of GDNF from the mesenchyme for survival, proliferation and migration of the neuroblasts (Gershon, 2010). However, the processes that support the neonatal ENS during innervation of the growing intestine, and then maintain the adult ENS are less clear. In particular, these processes may be involved in lessening the outcomes of damaging events, such as trauma or inflammatory damage, which is important to our understanding of disease

Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). T.Y. Han acknowledges a graduate scholarship from the Canadian Institutes of Health Research (CIHR) and the Canadian Association for Gastroenterology (CAG).

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