No neuronal loss, but alterations of the GDNF system in asymptomatic diverticulosis

<div><p>Background</p><p>Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to promote the survival and maintenance of neurons not only in the developing but also in the adult enteric nervous system. As diverticular disease (DD) is associated with reduced myenteric neurons, alterations of the GDNF system were studied in asymptomatic diverticulosis (diverticulosis) and DD.</p><p>Methods</p><p>Morphometric analysis for quantifying myenteric ganglia and neurons were assessed in colonic full-thickness sections of patients with diverticulosis and controls. Samples of tunica muscularis (TM) and laser-microdissected myenteric ganglia from patients with diverticulosis, DD and controls were analyzed for mRNA expression levels of GDNF, GFRA1, and RET by RT-qPCR. Myenteric protein expression of both receptors was quantified by fluorescence-immunohistochemistry of patients with diverticulosis, DD, and controls.</p><p>Results</p><p>Although no myenteric morphometric alterations were found in patients with diverticulosis, GDNF, GFRA1 and RET mRNA expression was down-regulated in the TM of patients with diverticulosis as well as DD. Furthermore GFRA1 and RET myenteric plexus mRNA expression of patients with diverticulosis and DD was down-regulated, whereas GDNF remained unaltered. Myenteric immunoreactivity of the receptors GFRα1 and RET was decreased in both asymptomatic diverticulosis and DD patients.</p><p>Conclusion</p><p>Our data provide evidence for an impaired GDNF system at gene and protein level not only in DD but also during early stages of diverticula formation. Thus, the results strengthen the idea of a disturbed GDNF-responsiveness as contributive factor for a primary enteric neuropathy involved in the pathogenesis and disturbed intestinal motility observed in DD.</p></div

Immunohistochemistry of RET and GFRα1 in colonic myenteric ganglia of patients with diverticulosis, DD, and controls.

<p>Myenteric immunopositive signals of RET in controls exhibited robust staining in neuronal somata and weaker signals in the surrounding neuropil (A). In patients with diverticulosis (B), the robust neuronal staining was slightly decreased, whereas the surrounding neuropil exhibited a clear reduction in immunopositive signals, when compared to controls. In patients with DD (C), not only the immunopositive RET signal of the neuropil is clearly decreased, but also the neuronal staining loses intensity. Myenteric immunopositive signals of GFRα1 in controls exhibited granular staining being confined to neuronal somata and a faint staining of the neuropil (D), which was weaker in patients with diverticulosis (E). In patients with DD some neurons exhibited strong GFRα1 positive signals (arrow), whereas others displayed only weak GFRα1 positive signals (arrowhead). The neuropil exhibited only weak immunopositive signals, when compared to controls (F). Bars = 20 μm</p

Morphometric analysis of the myenteric plexus in patients with diverticulosis compared to controls.

<p>Statistical comparison of ganglionic density (A) and mean ganglionic area (B) in patients with diverticulosis (d’osis) and controls revealed no significant differences as was neuronal density (C) and mean neuronal number (D). Data are graphically presented by box-whisker plots indicating the median (horizontal line), 50% of values (box) and 99% of values (whiskers).</p

mRNA expression of GDNF and its corresponding receptors RET and GFRA1 in the colonic tunica muscularis of patients with diverticulosis, DD, and controls.

<p>mRNA expression of GDNF (A), RET (B) and GFRA1 (C) is significantly decreased in both patients with diverticulosis (d’osis) and diverticular disease (DD), when compared to controls (c). mRNA levels are determined by qPCR, results of control group compared to DD was published before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171416#pone.0171416.ref017" target="_blank">17</a>]. Expression of target genes is normalized to mRNA expression of the house-keeping gene HPRT. Data are shown as mean +/- SEM, n = 9–12 per experimental group, *p<0.05 vs. control, **p<0.001 vs. control.</p