Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Summary

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Abstract

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Introduction

The gastrointestinal epithelial barrier plays a pivotal role in separating luminal antigens and pathogens from underlying tissue compartments¹. Epithelial injury and mucosal wounds seen in pathologic conditions such as inflammatory bowel disease (IBD), ischemia, or surgical injury are associated with clinical symptoms that include diarrhea, weight loss, blood in stool, and abdominal pain. In response to injury, epithelial cells migrate and proliferate to re-epithelialize and repair mucosal barrier defects. Resolution of inflammation and restitution of mucosal integrity are crucial to re-establishing intestinal mucosal homeostasis and function

Protocol

All animal experiments described were approved by the University of Michigan's Committee on the Use and Care of Animals.

1. Tissue harvest

1. Euthanize mice humanely using isoflurane anesthesia followed by cervical dislocation, in accordance with approved protocols. For all animal experiments, approval was obtained by a certified review board in accordance with the National and Institutional guidelines for animal handling.
2. Place the mouse on a dissecting pad in a supine .......

Discussion

Our Histological Colitis Score system constitutes a reliable tool to quantify tissue inflammation and damage in the intestine. This approach provides an improved understanding of the histopathological state of the whole organ without the bias of selecting small areas or incomplete sections. Among the critical steps to successfully execute this protocol are proper preparation of Swiss rolls that allow for analysis of at least 90% of the length of the colon; parallel orientation during paraffin embedding and sectioning to .......

Materials

Name	Company	Catalog Number	Comments
Aperio AT2 – High Volume, Digital Whole Slide Scanning	Leica Biosystems	Aperio AT2
Absorbent Underpads with waterproof moisture barrier	VWR International	56616-032
American Line 66-0089 Single Edge Blade, 100 per pkg	GT Midwest	TL5837
BD Luer-Lok Disposable Syringes without Needles	Fisher scientific	14-823-2A
Bonn Strabismus Scissors - ToughCut	Fine Science tools	11103-09
Bonn Strabismus Scissors - ToughCut	Fine Science tools	14084-09
Dumont #5 Forceps	Fine Science tools	11251-20
Formalin solution, neutral buffered, 10%	Sigma	HT501640-19L
HistoPrep 70% Ea	Fisherbran	70% denatured ethyl alcohol
ImageScope	Aperio	Version 12.3.3.5039	http://www.leicabiosystems.com/pathology-imaging/aperio-epathology/integrate/imagescope/
LeakBuster Specimen Containers: Sterile	Starplex Scientific	B120210
Phosphate-Buffere Saline, without calcium & magnesium	Corning	21-040-CV
Plastic Feeding tubes, 20 GA x 30 mm	Instech	FTP2030
PrecisionGlide Needle, Size: 20 G x 1 1/2 in	BD (Becton, Dickinson and Company)	305176
PrecisionGlide Needle, Size: 27 G x 1/2 in	BD (Becton, Dickinson and Company)	305109
Syringe, 10 ml	BD (Becton, Dickinson and Company)	302995
Unisette Tissue Cassettes	Simport	M505-2