Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Summary

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Abstract

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Introduction

Animal models are widely used in scientific research, and many non-invasive procedures exist to monitor disease activity and vitality, such as the quantification of body weight changes or the analysis of blood, urine, and feces. However, these are only indirect surrogate parameters that are also subject to inter-individual variability. They must frequently be complemented by post mortem analyses of tissue specimen, which prevents serial observation at repetitive time points and direct observation of physiological or pathological processes in vivo. Sophisticated small-animal imaging techniques have emerged, including cross sectional imaging, optical i....

Protocol

All animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz).

1. Materials and Experimental Setup

1. Animal care.
   1. Use sex- and age-matched mice of any DSS-susceptible strain (e.g., C57BL/6) at 20-25 g bodyweight.
   2. Plan at least five or more mice per experimental group and house the mice according to local animal care guidelines.
   3. Provide a standard rodent chow diet and autoclaved drinking water ad libitum.
   4. Remove the standard ....

Results

Assessment of Colitis:

DSS-induced colitis is a chemically induced murine model of intestinal inflammation that resembles human IBD and leads to weight loss, rectal bleeding, superficial ulceration, and mucosal damage in susceptible mice¹⁵. It is particularly useful to study the contribution of the innate immune system to the development of intestinal inflammation¹⁰

Discussion

Although medical imaging techniques have evolved rapidly in recent years, we are still limited in our ability to detect inflammatory processes or tumors, as well as other diseases, in their earliest stages of development. However, this is crucial to understanding tumor growth, invasion, or metastases development and cellular processes in the development of inflammatory disorders and degenerative, cardiovascular and immunological diseases. While traditional imaging techniques rely on physical or physiological parameters, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Alfalfa-free diet	Harlan Laboritories, Madison, USA	2014
Bepanthen eye ointment	Bayer, Leverkusen, Germany	80469764
Dextran sulphate sodium (DSS)	TdB Consulatancy, Uppsala, Sweden	DB001
Eosin	Sigma - Aldrich, Deisenhofen, Germany	E 4382
Ethylenediaminetetraacetic acid (EDTA)	Sigma - Aldrich, Deisenhofen, Germany	E 9884
Florene 100V/V	Abbott, Wiesbaden, Germany	B506
Haematoxylin	Sigma - Aldrich, Deisenhofen, Germany	HHS32-1L
O.C.T. Tissue Tek compound	Sakura, Zoeterwonde, Netherlands	4583	fixative for histological analyses
Phosphate buffered saline, PBS	Lonza, Verviers, Belgium	4629
Sodium Chloride 0,9%	Braun, Melsungen, Germany	5/12211095/0411
Sodium bicarbonate powder	Sigma Aldrich Deisenhofen, Germany	S5761
Standard diet	Altromin, Lage, Germany	1320
Tissue-Tek Cryomold	Sakura, Leiden, Netherlands	4566
Hemoccult (guaiac paper test)	Beckmann Coulter, Germany	3060
Biotin rat-anti-mouse anti-F4/80 antibody	Serotec, Oxford, UK	MCA497B
Biotin rat-anti-mouse anti-GR-1	BD Pharmingen, Heidelberg Germany	553125
Streptavidin-Alexa546	Molecular Probes, Darmstadt, Germany	S-11225	excitation/emission maximum:  556/573nm
Anti-CD11b rat-anti-mouse antibody TC	Calteg, Burlingame, USA	R2b06
Purified anti-mouse F4/80 antibody	BioLegend, London, UK	123102
DAPI	Sigma-Aldrich, Deisenhoffen, Germany	D9542
FITC-conjugated anti-Ly6C rat-anti-mouse antibody	BD Pharmingen, Heidelberg, Germany	553104
FACS buffer	BD Pharmingen, Heidelberg, Germany	342003
Cy7 NHS Ester	GE Healthcare Europe, Freiburg, Germany	PA17104
MPO ELISA	Immundiagnostik AG, Bensheim, Germany	K 6631B
Cy5.5 labeled anti-mouse F4/80 antibody	BioLegend, London, UK	123127	ready to use labelled Antibodies (alternative)
Anti-Mouse F4/80 Antigen PerCP-Cyanine5.5	eBioscience, Waltham, USA	45-4801-80	ready to use labelled Antibodies (alternative)
DMSO (Dimethyl sulfoxide)	Sigma-Aldrich, Deisenhoffen, Germany	67-68-5
Isoflurane	Sigma-Aldrich, Deisenhoffen, Germany	792632
Ethanol	Sigma-Aldrich, Deisenhoffen, Germany	64-17-5
Bovine Serum Albumins (BSA)	Sigma-Aldrich, Deisenhoffen, Germany	A4612
Tris Buffered Saline Solution (TBS)	Sigma-Aldrich, Deisenhoffen, Germany	SRE0032
Name	Company	Catalog Number	Comments
Equipment
FACS Calibur Flow Cytometry System	BD Biosciences GmbH, Heidelberg, Germany
FMT 2000 In Vivo Imaging System	PerkinElmer Inc., Waltham, MA, USA	FMT2000
True Quant 3.1 Imaging Analysis Software	PerkinElmer Inc., Waltham, MA, USA	included in FMT2000
Leica DMLB Fluorescent Microscope	Leica,  35578 Wetzlar, Germany	DMLB
Bandelin Sonopuls HD 2070	Bandelin, 12207 Berlin, Germany	HD 2070	ultrasonic homogenizer
Disposable scalpel No 10	Sigma-Aldrich, Deisenhoffen, Germany	Z692395-10EA
Metzenbaum scissors 14cm	Ehrhardt Medizinprodukte GmbH, Geislingen, Germany	22398330
luer lock syringe 5ml	Sigma-Aldrich, Deisenhoffen, Germany	Z248010
syringe needles	Sigma-Aldrich, Deisenhoffen, Germany	Z192368
Falcon Tube 50ml	BD Biosciences, Erembodegem, Belgium	352070