Decreased Intestinal Regeneration In Hirschsprung’s Disease
Purpose: Hirschsprung’s-associated enterocolitis is the leading cause of mortality in children with Hirschsprung’s disease. We hypothesize that impairment of stem cell regeneration is present in in the ganglionic colon contributing to Hirschsprung’s-associated enterocolitis.
Methods: Ednrb knockout mice (Ednrb-/-) with aganglionosis of rectum were studied. Following ethical approval (#47780), mice were sacrificed at postnatal day 21 (P21) and colon was harvested for analysis. Colonic epithelium was evaluated by H&E staining and the morphology of the distal ganglionic and the proximal ganglionic colon in Ednrb-/- mice (n=6) was compared to the corresponding colon of wildtype littermates (n=6). Proliferation marker Ki-67 and neuron marker Tuj1 were analysed by immunofluorescent staining. Quantification was performed by counting the number of Ki-67 positive stained cells per crypt by 3 blinded investigators. mRNA expression levels of inflammation and stem cell markers were studied by RT-qPCR. Statistical analysis was performed by t-tests.
Results: Distal ganglionic colon of Ednrb-/- mice showed greater damage (histological score 2.5) when compared with proximal ganglionic colon (score 1.0) and with corresponding colon of wildtype mice (score 0.5) (Fig. A, B). TNF- expression was upregulated in distal ganglionic colon of Ednrb-/- mice (Fig. C). Immunofluorescent staining of Ki-67 (Fig. D; white arrows indicate proliferative cells) and quantification of Ki-67 (Fig. E) were decreased in distal ganglionic colon compared with proximal ganglionic colon and with wildtype mice, indicating reduced epithelial proliferation. Neuron marker Tuj1 (green arrows indicate neurons) were present in both proximal and distal ganglionic colon. In addition, stem cell marker Lgr5 expression was decreased in the distal ganglionic colon of Ednrb-/- mice (Fig. F).
Conclusions: Epithelial damage occurs in the distal ganglionic colon of Hirschsprung’s (Ednrb-/-) mice. This injury is associated with decreased intestinal stem cell marker expression, epithelial proliferation, and epithelial regeneration. These novel findings support the occurrence of Hirschsprung’s-associated enterocolitis.