Name: fluorescence-mediated tomography for the detection and quantification of macrophage-related murine intestinal inflammation
Uploaded: 2017-12-15T12:30:00
Description: Watch this Scientific Journal Video about Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation at JoVE.com

We thank Ms. Sonja Dufentester, Ms. Elke Weber, and Mrs. Klaudia Niepagenk&#228;mper for the excellent technical assistance.

All animal experiments were approved by the Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen according to the German Animal Protection Law (Tierschutzgesetz).
1. Materials and Experimental Setup
<ol>
	<li>Animal care.
	<ol>
		<li>Use sex- and age-matched mice of any DSS-susceptible strain (e.g., C57BL/6) at 20-25 g bodyweight.</li>
		<li>Plan at least five or more mice per experimental group and house the mice according to local ani...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5A+apolipoprotein+A-I+(apoA-I)+mimetic+peptide+ameliorates+experimental+colitis+by+regulating+monocyte+infiltration.">The 5A apolipoprotein A-I (apoA-I) mimetic peptide ameliorates experimental colitis by regulating monocyte infiltration.</a> Br J Pharmacol. 173 (18), 2780-2792 (2016).</li><li>Hansch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+experimental+arthritis+with+near-infrared+fluorescence.">In vivo imaging of experimental arthritis with near-infrared fluorescence.</a> Arthritis Rheum. 50 (3), 961-967 (2004).</li><li>Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O., Yang, V. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vedolizumab+as+induction+and+maintenance+therapy+for+Crohn's+disease.">Vedolizumab as induction and maintenance therapy for Crohn's disease.</a> N Engl J Med. 369 (8), 711-721 (2013).</li><li>Vermeire, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etrolizumab+as+induction+therapy+for+ulcerative+colitis:+a+randomised,+controlled,+phase+2+trial.">Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial.</a> Lancet. 384 (9940), 309-318 (2014).</li><li>Coskun, M., Vermeire, S., Nielsen, O. 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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, ...

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 imaging, and endoscopy, which enables the direct visualization of these processes and also allows for repetitive analyses of the same animals1,2,3. Additionally, the possibility to repetitively monitor various states of disease in the same animal might decrease the number of animals needed, which might be desirable from an animal ethics point of view.
Several different optical imaging techniques exist for in vivo fluorescence imaging. Originally, confocal imaging was employed to study surface and subsurface fluorescent events4,5. Recently, however, tomographic systems that allow for quantitative three-dimensional tissue assessments have been developed6. This has been accomplished through the development of fluorescent probes that emit light in the near-infrared (NIR) spectrum, offering low absorption, sensitive detectors, and monochromatic light sources7. While traditional cross-sectioning imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), rely mostly on physical parameters and visualize morphology, optical imaging can provide additional information on underlying molecular processes using endogenous or exogenous fluorescent probes8.
Advances in molecular biology have helped to facilitate the generation of smart and targeted fluorescent molecular probes for an increasing number of targets. For example, receptor-mediated uptake and distribution in a given target area can be visualized using carbocyanine derivative-labeled antibodies9. The abundance of available antibodies, which can be labeled to function as specific tracers in otherwise inaccessible areas of the body, provides unprecedented insights into molecular and cellular processes in models of tumorigenesis and neurodegenerative, cardiovascular, immunologic, and inflammatory diseases7.
In this study, we describe the use of fluorescence-mediated tomography in a murine model of colitis. Dextran sodium sulfate (DSS)-induced colitis is a standard chemically induced mouse model of intestinal inflammation that resembles inflammatory bowel disease (IBD)10. It is particularly useful to assess the contribution of the innate immune system to the development of gut inflammation11. Since the recruitment, activation, and infiltration of monocytes and macrophages represent crucial steps in the pathogenesis of IBD, visualization of their recruitment and the kinetics of infiltration are essential to monitoring, for example, the effect of potential therapeutic substances in a preclinical setting12. We describe the induction of DSS colitis and demonstrate the tomography-mediated characterization of macrophage infiltration into the gut mucosa using fluorescence molecular tomography for the specific visualization of the monocyte/macrophage marker F4/8013. Additionally, we illustrate auxiliary and supplemental procedures, such as antibody labelling; the experimental setup; and analysis and interpretation of the obtained images, in correlation with conventional readouts such as disease activity indices, flow cytometry and histological analysis, and immunohistochemistry. We discuss limitations of this technique and comparisons to other imaging modalities.

<table border="0" cellpadding="0" cellspacing="0" width="1601">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Reagents</td>
			<td style="width:423px;"></td>
			<td style="width:320px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alfalfa-free diet</td>
			<td>Harlan Laboritories, Madison, USA</td>
			<td style="width:320px;">2014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bepanthen eye ointment</td>
			<td style="width:423px;">Bayer, Leverkusen, Germany</td>
			<td style="width:320px;">80469764</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulphate sodium (DSS)</td>
			<td>TdB Consulatancy, Uppsala, Sweden</td>
			<td>DB001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Eosin</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 4382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Ethylenediaminetetraacetic acid (EDTA)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Florene 100V/V</td>
			<td>Abbott, Wiesbaden, Germany</td>
			<td style="width:320px;">B506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Haematoxylin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">HHS32-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">O.C.T. Tissue Tek compound&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sakura, Zoeterwonde, Netherlands</td>
			<td style="width:320px;">4583</td>
			<td>fixative for histological analyses</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Phosphate buffered saline, PBS</td>
			<td style="width:423px;">Lonza, Verviers, Belgium</td>
			<td style="width:320px;">4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride 0,9%</td>
			<td>Braun, Melsungen, Germany</td>
			<td style="width:320px;">5/12211095/0411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate powder</td>
			<td>Sigma Aldrich Deisenhofen, Germany</td>
			<td style="width:320px;">S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Standard diet</td>
			<td>Altromin, Lage, Germany</td>
			<td style="width:320px;">1320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold</td>
			<td>Sakura, Leiden, Netherlands</td>
			<td style="width:320px;">4566</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemoccult (guaiac paper test)</td>
			<td>Beckmann Coulter, Germany</td>
			<td style="width:320px;">3060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin rat-anti-mouse anti-F4/80 antibody</td>
			<td>Serotec, Oxford, UK</td>
			<td style="width:320px;">MCA497B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotin rat-anti-mouse anti-GR-1&#160;</td>
			<td>BD Pharmingen, Heidelberg Germany</td>
			<td>553125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-Alexa546</td>
			<td>Molecular Probes, Darmstadt, Germany</td>
			<td style="width:320px;">S-11225</td>
			<td>excitation/emission maximum:&#160; 556/573nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD11b rat-anti-mouse antibody TC</td>
			<td>Calteg, Burlingame, USA</td>
			<td style="width:320px;">R2b06</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Purified anti-mouse F4/80 antibody</td>
			<td>BioLegend, London, UK</td>
			<td style="width:320px;">123102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td style="width:320px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC-conjugated anti-Ly6C rat-anti-mouse antibody</td>
			<td>BD Pharmingen, Heidelberg, Germany</td>
			<td>553104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS buffer</td>
			<td>BD Pharmingen, Heidelberg, Germany</td>
			<td style="width:320px;">342003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy7 NHS Ester</td>
			<td>GE Healthcare&#160;Europe, Freiburg, Germany</td>
			<td style="width:320px;">PA17104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MPO ELISA</td>
			<td>Immundiagnostik AG, Bensheim, Germany</td>
			<td>K 6631B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy5.5 labeled anti-mouse F4/80 antibody</td>
			<td>BioLegend, London, UK</td>
			<td>123127</td>
			<td>ready to use labelled Antibodies (alternative)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Mouse F4/80 Antigen PerCP-Cyanine5.5</td>
			<td>eBioscience, Waltham, USA</td>
			<td>45-4801-80</td>
			<td>ready to use labelled Antibodies (alternative)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO (Dimethyl sulfoxide)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Isoflurane</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumins (BSA)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>A4612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Buffered Saline Solution (TBS)</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>SRE0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Name</td>
			<td style="width:423px;">Company</td>
			<td style="width:320px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS Calibur Flow Cytometry System</td>
			<td>BD Biosciences GmbH, Heidelberg, Germany</td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FMT 2000 In Vivo Imaging System</td>
			<td>PerkinElmer Inc., Waltham, MA, USA</td>
			<td style="width:320px;">FMT2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">True Quant 3.1 Imaging Analysis Software</td>
			<td>PerkinElmer Inc., Waltham, MA, USA</td>
			<td style="width:320px;">included in FMT2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica DMLB Fluorescent Microscope</td>
			<td>Leica,&#160; 35578 Wetzlar, Germany&#160;</td>
			<td>DMLB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bandelin Sonopuls HD 2070</td>
			<td>Bandelin, 12207 Berlin, Germany</td>
			<td style="width:320px;">HD 2070</td>
			<td>ultrasonic homogenizer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Disposable scalpel No 10</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>Z692395-10EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Metzenbaum scissors 14cm</td>
			<td>Ehrhardt Medizinprodukte GmbH, Geislingen, Germany</td>
			<td>22398330</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">luer lock syringe 5ml</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td style="width:320px;">Z248010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">syringe needles</td>
			<td>Sigma-Aldrich, Deisenhoffen, Germany</td>
			<td>Z192368&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Tube 50ml</td>
			<td>BD Biosciences, Erembodegem, Belgium</td>
			<td style="width:320px;">352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-mediated tomography for the detection and quantification of macrophage-related murine intestinal inflammation

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.

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</sup...

Watch this Scientific Journal Video about Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation at JoVE.com

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

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.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

The overall goal of this target-specific in vivo imaging approach is to visualize molecular markers of disease activity in inflammatory bowel disease. This method can help answering key questions in the field of inflammatory bowel disease, and related therapeutic research about the influence of experimental therapeutics or specific cellular events on inflammation. The main advantage of this technique is that the disease activity can be monitored longitudinally and noninvasively.Though this method can provide insight into inflammatory disorders, it can also be applied to study other diseases, such as cancer or immune-related diseases. To induce colitis, fill the drinking supply of the experimental mice with two grams of dextran sodium sulfate, or DSS, dissolved in 100 milliliters of autoclaved drinking water, estimating five milliliters of water per mouse per day. 24 hours before scanning, load the antibody cocktail of interest into a sterile syringe protected from light and inject the antibodies intravenously into the tail veins of the experimental animals.On the day of the experiment, use an electric razor to shave the fur in the abdominal region of the experimental animals to minimize light reflection and absorption. Using a tissue-mimicking phantom of defined thickness and absorption characteristics, fill the phantom with a specific volume of the antibody solution of interest and measure the fluorescence on the FMT device. Then, on a veterinary fluorescence-mediated tomography, or FMT device for small animals, click New Study and include the relevant tracers in the study description, including the imaging parameters and doses.Create study groups according to the experimental design, equipping each group with the appropriate number of animals. When the system has been calibrated for the tracer constructs, place the first anesthetized mouse in the supine position into a 42 degrees Celsius heatable examination cassette. Insert the cassette into the imaging system and select the appropriate sample from the corresponding study group.Select the administered tracer to ensure that the values of the tracer concentration are properly calculated, and acquire the fluorescence reflectance image at the appropriate wavelength for the scan. Click Acquire Image to outline the scan field and confirm that the scan field appears as an overlay on the fluorescence reflectance image. Adjust the scan field until it surrounds the region of interest, taking care to avoid error or areas of remaining fur.Depending on the image target, select a scan field resolution to set the number of image data points within the scan field. Then click Scan to begin the image acquisition at the selected wavelength. At the end of the scan, remove the animal from the cassette to allow the animal to fully recover with monitoring before returning to its cage.The FMT can then be repeated at various time points as experimentally appropriate. To analyze the acquired images, open the appropriate imaging analysis software and select the raw data for the first scan. Using the reconstruction tool, create 3D maps of the fluorescence distribution.When all of the scans have been added to the appropriate reconstruction dataset, open the first dataset of interest. All of the scans performed for the selected individual experimental animal will be shown. Select the appropriate scan and click Load.A 3D reconstruction of the tracer distribution will appear as an overlay of the initially acquired fluorescence reflectance image. Next, identify the foci of unspecific label accumulation on the reconstructed 3D maps and use the measuring tool to select the region of interest. The software will then generate a fluorescence intensity for the region of interest, as well as picomolar amount measurements of the tracer for which the scan was calibrated.In this experiment, a fluorescence conjugated antibody against murine F4/80 was employed to directly visualize the activated macrophages. A significantly elevated accumulation of fluorescent tracer was measured by fluorescence-mediated tomography in the colons of the colitic mice compared to the non-colitic control animals, indicating an increase in monocyte infiltration and differentiation into active macrophages in the colitic animals. Conversely, the application of a non-specific fluorescence conjugated IgG antibody did not illicit a detectable fluorescence response in the abdomen or intestine in either the colitic or non-colitic control group, demonstrating the specificity of the probe-target interaction of the F4/80 antibody.Ex vivo measurements of F4/80-directed tracer accumulation in explanted colons further verify the colonic origin of the in vivo detected F4/80 tracer signal accumulation. Indeed, calculated amounts of bound antibody correlate well with histologically determined numbers of infiltrating macrophages, confirming that in vivo imaging with specific tracers can be indicative of local disease activity. Once mastered, this can be performed and analyzed within a couple of minutes if it is performed properly.The preparation steps, including the production of specific antibody-based tracers, typically take two to three days. When planning an experimental procedure such as this one, it is important to include appropriate and reliable controls, both for the antibody and the animal disease model. Following this procedure, other methods, like colonoscopy, or flow cytometry can be performed to answer additional questions and to validate and quantify the data.

Research

JoVE Journal

Medicine

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