Investigating Intestinal Inflammation in DSS-induced Model of IBD

Summary

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Abstract

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.¹ Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.²

There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.³ Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.^4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.⁶

The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.⁷ Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.⁸

In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Protocol

1. Murine model of DSS-induced acute colitis

1. Add dextran sulphate sodium (DSS) to autoclaved drinking water to the desired final concentration (1-5%) (wt/vol) (i.e. To make a 5% DSS solution, add 25 g of DSS powder to 500 mL of autoclaved water). Stock solution can be left at room temperature for up to one week or at 4°C until use.
2. In a biosafety hood, pour stock DSS solution into 50 mL Falcon tubes (one needed per cage). Keep the stock solution to refill tubes when needed.
3. Replace drinking water in each mouse cage with the DSS solution (in the 50 mL Falcon tubes) (The duration will depend on the DSS regimen that is used. For example, u....

Discussion

DSS colitis is a widely used chemically induced model of intestinal inflammation. In this model, mice are given drinking water supplemented with DSS, which is thought to be toxic to gut epithelial cells and disrupt the integrity of the mucosal barrier.¹⁰ Administration of DSS induces an acute colitis that is characterized by loose stool, fecal bleeding, and infiltration with granulocytes.¹⁰ During DSS administration, colitis is usually associated with significant weight loss and presence of blood in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Eppendorf Safe-Lock Microcentrifuge Tube (2mL)	Eppendorf	0030 120.094
Biotek EL808 Absorbance plate reader	BioTek	EL808
Dextran sulphate sodium salt reagent grade (molecular weight 36,000-50,000 Da)	MP Biomedicals	160110
Gen5 (software)	BioTek	Version 1.10.8
Hexadecyltrimethylammonium bromide (HTAB)	Sigma-Aldrich	H5882-100G
Hydrogen peroxide, 30 wt.% solution in water	Sigma-Aldrich	216763	Store at 2-8°C
Microtest plate 96-well flat bottom	Sarstedt Ltd	82.1581	For single use only
o-Dianisidine	Sigma-Aldrich	D-3252	Light sensitive. Store at 2-8°C
Potassium phosphate, dibasic	Caledon	6620-1
Potassium phosphate, monobasic	EMD Millipore	PX1565-1
Protease inhibitor cocktail	Sigma-Aldrich	P8340	Store at -20°C
Triton X-100	Sigma-Aldrich	T8787
Tungsten Carbide beads for Tissue Lyser II	Qiagen	69997