Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Summary

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Abstract

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.^1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.³ The inflammation is characterized by liberation of proinflammatory mediators such as IL-6⁴ or IL-1β or inhibitory neurotransmitters like nitric oxide (NO).⁵ Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.² Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.⁶

In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Protocol

1. Animals

Male C57Bl6/J mice with a mean weight of 25 g were obtained from Harlan Winkelmann (Borchen, Germany). All experiments were performed in accordance with the federal law regarding the protection of animals. The principles of laboratory animal care were followed. Animals were maintained on a 12-hr light/dark cycle and provided with commercially available rodent chow and tap water ad libitum. Animals should be housed at least 7 days after arrival in your animal facility before experiments. Note that all experiments can be easily adapted to rats or other species and with slight modifications into large animal models.....

Discussion

A comparative study analyzing different levels of IM (small bowel eventration for 10 min versus gently inspection of the small bowel using two cotton applicators versus IM with evacuation of small bowel content into the cecum) revealed a correlation between the extent of surgical manipulation and POI. Compared with the other groups IM showed the highest amount of leukocytes in the ME (PNMs, macrophages and mast cells) resulting in prolonged and severe POI.⁸.

When establishing the mo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethilon 5/0	Ethicon	1666H
Cotton applicators	MaiMed	71010
arterial catheter	Vygon	115-798
96 well plate (black)	Greiner Bio One	655096
glass ball (2 mm diameter)	Worf	available on request
Insect pins	Fine Science Tools	26000-25
Hanker Yates Reagent	Polyscience	560694
Aquatek	Merck	HC109850
DMEM	Lonza	BE12-604 F
FITC-dextran (MW 70000)	Sigma Aldrich	46945-500MG-F