Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease

Abstract

Background Hirschsprung’s disease (HSCR) is a congenital condition in which enteric ganglia, formed from neural crest cells (NCC), are absent from the terminal bowel. Dysmotility and constipation are common features of HSCR that persist following surgical intervention. This persistence suggests that the portion of the colon that remains post-operatively is not able to support normal bowel function. To elucidate the defects that underlie this condition, we utilized a murine model of HSCR. Methods Mice with NCC specific deletion of Ednrb, were used to measure the neuronal density and neurotransmitter expression in ganglia. Key Results At the site located proximal to the aganglionic region of P21 Ednrb null mice, the neuronal density is significantly decreased and the expression of neurotransmitters is altered compared to het animals. The ganglia in this colonic region are smaller and more isolated while the size of neuronal cell bodies is increased. The percentage of neurons expressing neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) is significantly increased in Ednrb nulls. Conversely, the percentage of choline acetyltransferase (ChAT) expressing neurons is decreased, while Substance P is unchanged between the two genotypes. These changes are limited to the colon and are not detected in the ileum. Conclusions & Inferences We demonstrate changes in neuronal density and alterations in the balance of expression of neurotransmitters in the colon proximal to the aganglionic region in Ednrb null mice. The reduced neuronal density and complementary changes in nNOS and ChAT expression may account for the dysmotility seen in HSCR.

Bowel dysfunction following pullthrough surgery is associated with an overabundance of nitrergic neurons in Hirschsprung disease

“…Studies in mice have suggested that the extent of neuronal subtype imbalance also correlates with the length of aganglionosis [8]; however, we found no correlation between the proportion of NOS neurons and the length of bowel resected. In addition, studies have shown that disturbances in NOS expression are greater in older HD mice [9], but we found no similar correlation between NOS and age in our patients. As in prior studies [22], we also found no correlation between constipation and age at surgery.…”

“…NOS+ neurons are one of the first subtypes to appear in embryonic mouse colon [20], and failure of neuronal maturation to progress normally may result in an overabundance of NOS+ neurons. In previous studies in mouse models of HD, neuronal subtype imbalance was found to be limited to the mid and distal colon and was normal in proximal colon and small bowel [8, 9]. The extent of neuronal abnormality in HD patients is not yet known and warrants further study, as this knowledge is essential to determine the appropriate length of resection and to predict functional outcome after pullthrough.…”

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung’s disease

“…However, there is increasing evidence that ENS abnormalities in HSCR extend beyond the aganglionic region, and the concept is emerging that the ganglionated bowel in HSCR may in fact be abnormal [39,40]. In addition, genes that regulate ENS formation may also be involved in development of mucosal immunity [24].…”

Appearance of cholinergic myenteric neurons during enteric nervous system development: comparison of different ChAT fluorescent mouse reporter lines

“…These data are in contrast to the alterations in neurotransmitter expression observed in mice where neuronal density has been genetically modified. Our conditional mouse knockout model of Hirschsprung’s disease (HSCR) showed an increase in the proportion of NOS expressing neurons and a decrease in the percentage of cholinergic neurons (15). These changes were correlated with reduced neuronal density in the colon just proximal to the aganglionic segment.…”

“…They were then washed in PBS (3 × 1 hour), and finally placed into blocking solution containing the secondary antibodies as previously described [(Supp. Table 1; (15)].…”