Pan-enteric neuropathy and dysmotility are present in a mouse model of short-segment Hirschsprung disease and may contribute to post-pullthrough morbidity

Abstract

Purpose: Hirschsprung disease (HSCR) is characterized by distal intestinal aganglionosis. While surgery is lifesaving, gastrointestinal (GI) motility disorders persist in many patients. Our objective was to determine whether enteric nervous system (ENS) abnormalities exist in the ganglionated portions of the GI tract far proximal to the aganglionic region and whether these are associated with GI dysmotility.

Methods: Using Ednrb-null mice, a model of HSCR, immunohistochemical analysis was performed to evaluate quantitatively ENS structure in proximal colon, small intestine, and stomach. Gastric emptying and intestinal transit were measured in vivo and small and large bowel contractility was assessed by spatiotemporal mapping ex vivo.

Results: Proximal colon of HSCR mice had smaller ganglia and decreased neuronal fiber density, along with a marked reduction in migrating motor complexes. The distal small intestine exhibited significantly fewer ganglia and decreased neuronal fiber density, and this was associated with delayed small intestinal transit time. Finally, in the stomach of HSCR mice, enteric neuronal packing density was increased and gastric emptying was faster.

Conclusions: ENS abnormalities and motility defects are present throughout the ganglionated portions of the GI tract in Ednrb-deficient mice. This may explain the GI morbidity that often occurs following pull-through surgery for HSCR.

Keywords: Dysmotility; Ednrb; Enteric nervous system; Hirschsprung disease.

Figure 1.. Ednrb KO mice possess ENS abnormalities in the ganglionated proximal colon.

Cross sections of the colon were stained with neuronal (Hu) and glial (Nestin) antibodies (a,b) and show that KO mice have a similar density of ganglia (c), but the ganglia are smaller in size (d). Wholemount preparations stained with neuronal antibodies, Tuj1 and Hu (e, f), reveal that neuronal cell density in the myenteric plexus is similar between the two groups (g), but KO mice have lower nerve fiber density (h). No difference in neuronal packing density was seen between the two groups (i).

Figure 2.. Proximal colon of Ednrb KO mice has markedly abnormal contractile function.

Spatiotemporal mapping of colonic contractility shows normal propagating CMMCs in WT mice (a, arrows), but these are nearly absent in KO (b,c).

Figure 3.. ENS abnormalities are present in the distal small intestine of Ednrb KO.

Cross sections of the small intestine were stained with the neuronal antibody, Hu (a,b), and reveal lower ganglion density in KO mice (c), but with similar ganglion size (d). Wholemount preparations stained with neuronal antibodies, Tuj1 and Hu (e, f), show that neuronal cell density in the myenteric plexus is similar between the two groups (g), but KO mice have lower nerve fiber density (h) and increased neuronal packing density (i).

Figure 4.. Distal small intestinal transit is delayed in Ednrb KO mice.

Spatiotemporal mapping of distal small intestine (a-c) shows that the frequency (d), amplitude (e), and AUC (f) of segmentation contractions are not altered in Ednrb KO mice. In vivo analysis of intestinal transit, however, shows a significant delay at 40 minutes, but not 20 minutes, in KO mice (g), corresponding with delayed transit in the distal half of the small intestine.

Figure 5.. Ednrb KO mice exhibit ENS abnormalities and motor dysfunction in the gastric antrum.

Wholemount preparations of gastric myenteric plexus were stained with neuronal antibodies, Tuj1 and Hu (a, b). Quantitative analysis demonstrates similar density of neuronal cell bodies (c) and neuronal fibers (d) between the two groups, with a higher neuronal packing density in KO mice (e). Gastric emptying was significantly faster in the KO group (f).