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Summary

Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

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

Published: February 14th, 2021

DOI:

10.3791/62135

1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University

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.

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.

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

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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 .......

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To illustrate the reliability of this Histological Colitis Score Analysis in the context of mucosal damage after DSS challenge and subsequent recovery from colitis, we administered 2.5% DSS in the drinking water of eight 10 weeks old male C57BL6 wild type mice for 5 days followed by a recovery period with regular water for 5 days. There was no change in body weight during the acute administration of DSS, from day 0 to 5 (Figure 4A). Body weight dramatically d.......

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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 .......

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The authors wish to acknowledge support from NIH funding DK055679, DK089763, DK079392, DK061739, DK072564 and the University of Michigan Pathology Slide Scanning Services.

....

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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

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