Murine model of Hirschsprung-associated enterocolitis II: Surgical correction of aganglionosis does not eliminate enterocolitis

Abstract

Background: Hirschsprung disease (HD) results from aganglionosis of the colon, is linked to acute and chronic enterocolitis (known as Hirschsprung-associated enterocolitis) despite successful corrective surgery, and can lead to bacteremia and even death. The genetic and molecular mechanisms underlying these disorders are largely unknown.

Methods: We developed a microsurgical corrective pull-through procedure in mice, and applied that to Ednrb(-/-) mice, which manifest aganglionic megacolon that is very similar to HD. Wild-type littermates (Ednrb(+/+)) also underwent identical surgery. At prespecified time points postoperatively, mice were sacrificed, and histopathologic analyses of intestinal inflammation were performed. Mice of both genotypes were sacrificed after the postoperative recovery period to determine if corrective surgery itself caused inflammation. Stooling patterns were assessed as well to determine if intestinal function normalized after surgery.

Results: There was no difference in histopathological enterocolitis scores after recovery from surgery. Stooling patterns in Ednrb(-/-) and Ednrb(+/+) mice were similar postoperatively, suggesting normalization of intestinal function. However, with time, approximately 40% of Ednrb(-/-) mice developed clinical illness consistent with enterocolitis. No control (Ednrb(+/+)) mice developed clinical enterocolitis. Histopathological enterocolitis scores in the 40% of Ednrb(-/-) mice that developed clinical enterocolitis postoperatively were significantly worse than those of healthy postoperative Ednrb(-/-) mice. In contrast, none of the Ednrb(+/+) control mice exhibited postoperative long-term inflammation.

Conclusions: Microsurgical pull-through operation in Ednrb(-/-) mice produces a mouse model that closely resembles key features of Hirschsprung-associated enterocolitis, enabling controlled study of genetic and molecular mechanisms in Ednrb(-/-) mice and other genotypes that produce similar phenotypes.

Fig. 1

Murine colon pullthrough operation. A, Resection of dilated megacolon and aganglionic segment. B, Preparation of pullthrough segment. C, Completed pullthrough operation with coloanal anastomosis.

Fig. 2

Pathologic segments evaluated after pullthrough operation. Segment 1 included the coloanal anastomosis and approximately 1 cm of distal pullthrough segment. Segment 2 included 1 cm of colon proximal to the ileum, and segment 3 included 1 cm of terminal ileum.

Fig. 3

Stooling frequency after pullthrough operation. Four Ednrb^+/+ and 4 Ednrb^−/− mice, 4 weeks after pullthrough operation had stooling frequency assessed over a 5-day period. There was no significant difference in numbers of stools per day using Wilcoxon rank sum test.

Fig. 4

Enterocolitis scores of post-pullthrough mice. Enterocolitis scores (inflammation, depth and total) were compared between each group of Ednrb^+/+ and Ednrb^−/− mice. No significant difference between mean enterocolitis scores was found.

Fig. 5

Enterocolitis scores of healthy and clinically ill postpullthrough Ednrb^−/− mice. Enterocolitis scores (inflammation, depth and total) were compared between healthy and clinically ill Ednrb^−/− mice. There was a statistically significant difference in the inflammation, depth, and total enterocolitis scores between the healthy and ill mice (Wilcoxon rank sum test P < .05).

Fig. 6

Percent of the healthy animals after the pullthrough operation of each genotype. There was a statistically significant difference between (Fischer exact test, P < .05).