Murine model of Hirschsprung-associated enterocolitis. I: Phenotypic characterization with development of a histopathologic grading system

Abstract

Purpose The aim of the study was to characterize enterocolitis in the Ednrb-null (Ednrb−/−) mouse with aganglionosis of the colon and to develop and validate a semiquantitative histopathologic grading system to assess enterocolitis. Methods We isolated colon and ileal specimens of Ednrb−/− and control mice (Ednrb+/+) and performed histochemical staining (H&E) on tissue sections. After establishing inflammation grading criteria, 2 blinded pathologists independently assessed the severity and depth of inflammation of proximal colon segments on 2 separate occasions. Interclass correlations (ICCs) and coefficient of variation (CV) were calculated to determine interrater and intrarater agreement. We then prospectively applied the enterocolitis grading system to Ednrb−/− mice that became clinically ill. A cohort of Ednrb−/− mice were observed until they developed clinical illness, at which time they were euthanized and had multiple organ homogenates cultured for bacteria, and colon and small bowel were histopathologically graded for enterocolitis. Spearman’s rank correlations comparing enterocolitis scores with level of bacteremia were performed. Results Intra- and interrater ICCs of the histologic scoring system were satisfactory (0.61 and 0.94, respectively), as were intra- and interrater CVs (18% and 9%, respectively). Of the Ednrb−/− mice, 65% developed bacteremia. Those with bacteremia had significantly higher enterocolitis scores than those without bacteremia (P < .01). Ednrb−/− mice that developed bacteremia showed a strong positive correlation between total enterocolitis scores and number of bacterial colony forming units in peritoneal lavage, liver, kidney, and aerobic spleen. Conclusions The Ednrb−/− mouse with aganglionosis develops enterocolitis and has features similar to Hirschsprung-associated enterocolitis in humans. Our grading system is a reliable way to assess enterocolitis. By performing microsurgical pull-through, we can now perform controlled, hypothesis-driven, mechanistic studies to evaluate etiologic factors affecting enterocolitis in the Ednrb−/− mouse.

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

“…In keeping with published results, we noted only the occasional presence of neutrophils in some of the P21 EdnrB -null colons, but found a robust inflammatory cell infiltrate present at P26 [37]. As with the survival data, it is likely that there is a spectrum of colonic inflammation between these two points.…”

“…Previous studies of the naturally occurring EdnrB mutation (piebald lethal mouse strain) and the conventional EdnrB deletion have documented the development of enterocolitis prior to death and a mean survival time between 3–4 weeks of age [36,37]. In order to confirm the utility of the neural crest-conditional deletion EdnrB -null model in the study of HAEC, animals were followed until moribund and survival recorded (Figure 1A).…”

Altered Goblet Cell Differentiation and Surface Mucus Properties in Hirschsprung Disease

“…Ednrb −/− mice die at age 3–5 weeks from either distal intestinal obstruction or HAEC-associated sepsis [25]. HAEC onset occurs typically soon after weaning (P21), with rapid progression thereafter.…”

821 Altered Goblet Cell Differentiation and Surface Mucus Properties in Hirschsprung Disease

“…Endothelin receptor B (Ednrb) mutant mice are a well-described model of colorectal aganglionosis that exhibit many of the features of human HSCR including megacolon and development of HAEC [24], [25]. Ednrb 2/2 mice die at age 3-5 weeks from either distal intestinal obstruction or HAEC-associated sepsis [25].…”

“…Ednrb 2/2 mice die at age 3-5 weeks from either distal intestinal obstruction or HAEC-associated sepsis [25]. HAEC onset occurs typically soon after weaning (P21), with rapid progression thereafter.…”