18 Mast Cell Dependent Inflammatory Responses Mediate Ileus in a Mouse Model of Surgical Bowel Manipulation

Abstract

Hirschsprung's disease (HD) is a congenital disorder characterized by aganglionosis in the distal intestine. The zebrafish mutant, lessen (lsn), has HD phenotypic characteristics. We use the lsn mutant as an experimental model to unravel underlying developmental mechanisms for HD. This study aims to compare the neurochemical content of enteric neurons and gastrointestinal (GI)-motility patterns between wild-type zebrafish and lsn mutants to further validate this mutant as a suitable model for HD research. Immunofluorescence staining was used to detect specific neurochemical markers from 3 to 6 days post-fertilization (dpf) in the proximal (PI), mid (MI) and distal intestine (DI) of both wild-type and mutant embryos. Contractile activity of the GI-tract was filmed and the frequency and direction of contractions in each intestinal region were analyzed. Both mutant and wild-type embryos showed an increase in enteric neuron numbers over time. In mutants, the number of enteric neurons at each embryonic stage was significantly reduced in DI (absent at 3dpf) and MI, but less in PI. The proportion of nitrergic neurons was significantly reduced in all regions at 3dpf, but nearly unaffected in PI and MI at 4 and 5dpf. In mutants, serotonin, calretinin and calbindin showed a delayed expression and a decrease in both number and proportion at all points of time and in each intestinal region. In both mutant and wild-type embryos, galanin, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) were only detected in nerve fibers. In wild-type, VIP/PACAP-positive and galaninpositive fibers were present at all time points and in each region. Mutants only showed a few immunoreactive fibers at 3dpf in PI, while at 4dpf immunostained fibers appeared in MI and at 5dpf in DI, showing a delayed expression of these neuropeptides. GI-contractility analyses revealed that wild-type animals have defined motility patterns by 4dpf. When comparing motility patterns, the PI showed little change in when comparing wild-type to mutants. In contrast, we observed a decreased contractility in the MI and DI of mutants. The present study reveals abnormalities in the number and proportions of neurons expressing various neurochemical markers in the lsn mutant. These results are similar to previously reported data in the intestine proximal to the aganglionic segment and in the aganglionic segment of the lethal spotted mutant mice, an experimental HD model. Furthermore, in lessen GI-contractility is significantly perturbed inMI and DI. So, this study supports previous studies indicating that the zebrafish mutant lessen is a suitable model for HD research.