W. N. El-Nachef1, M. K. Collins1, T. C. Grikscheit1,2 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2University Of Southern California,Keck School Of Medicine,Los Angeles, CA, USA
Introduction:
Functional bowel disorders such as colonic pseudo-obstruction, idiopathic megacolon, Hirschsprung's disease, and postsurgical ileus are incompletely understood. Models of functional bowel obstruction will assist in exploring the pathophysiology of these entities; however, previous models rely on mechanical means to obstruct bowel. Benzalkonium chloride (BAC) was first shown in the 1970s to ablate the myenteric plexus in rats when applied to the serosal surface of bowel. However, we have found this approach to be limited by the need to perform laparotomy, the wide spread and difficult to control field of exposure, and the difficulty to reproduce nonlethal obstruction in mice. Here, we describe a non-invasive protocol in which BAC is applied to the luminal surface of murine colon, leading to ablation of the submucosal plexus and a dramatic functional obstruction.
Methods:
Male C57/BL6 mice aged 2 months were selected to receive PBS, 5% BAC, or 10% BAC per rectum, with 3 mice per group. Extra-small craft cotton swabs were soaked in the respective solution and then inserted per rectum until the bottom-most part of the cotton applicator was just beyond the anus and then left in place for 4 minutes. The cotton swab was then removed with gentle counter-pressure to prevent prolapse. This was repeated for a total of 4 treatments, with the last cotton swab duration being 3 minutes, for a total of 15 minutes of exposure.
The mice were observed daily and sacrificed on post-exposure day 5. Colons were resected and prepared into paraffin blocks for histologic analysis with hematoxylin and eosin (H&E) and immunofluorescent (IF) staining with antibodies to the pan-neuronal marker TUJ-1 and smooth muscle actin.
Results:
At time of resection, all PBS-treated colons appeared phenotypically normal, with discrete fecal pellets visible within the lumen. Colons treated with 10% BAC were uniformly and massively obstructed with rigid-dilatation due to impacted feces; H&E revealed an intact epithelium but an enlarged submucosa with cellular infiltrate, and IF staining revealed an absence of TUJ-1 in the submucosa but sparing of the myenteric plexus. Colons treated with 5% BAC had less severe dilatation, a smaller submucosal infiltrate, and scant TUJ-1 staining in the submucosa. PBS-treated mice displayed normal histology and IF staining patterns.
Conclusion:
We have demonstrated that BAC can be applied to the luminal surface of intestine to effect a functional obstruction, the severity of which appears to be dose dependent. This obstructive phenotype is accompanied by the decreased/absent visualization of submucosal neurons on IF staining, though myenteric neurons appear intact. This suggests that perturbations to the submucosal plexus alone can result in functional obstructive disorders. This protocol is non-invasive, not dependent on mechanical obstruction, and technically simple; thus, it can be easily replicated to model functional bowel disorders.