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 imaging, and endoscopy, which enables the direct visualization of these processes and also allows for repetitive analyses of the same animals^1,2,3. 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 events^4,5. 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.

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 chow and replace it with alfalfa-free chow at least three days prior to scanning to reduce endoluminal auto-fluorescence.
2. Induction of acute DSS-induced colitis.
   1. Dissolve 2 g of DSS (molecular weight ~40,000 Da) in 100 mL of autoclaved drinking water to obtain a 2% (w/v solution).
   2. Fill the drinking supply of the mice exclusively with the DSS solution and estimate 5 mL of liquid per mouse per day. Provide the same drinking water without DSS to the control mice¹⁰.
      NOTE: Monitor the mice daily until the end of the experiment. Euthanize the mice that lose greater than 20% of their initial body weight or that become moribund (i.e., persistently hunched posture, decreased movement, labored breathing, markedly erect coat) according to local applicable guidelines on animal welfare.
3. Preparation of fluorescence-mediated tomography.
   1. Label the desired antibody (e.g., rat anti-mouse F4/80) with fluorescence dye (e.g., Cyanine7, λ_excitation: 750 nm, λ_emission: 776 nm) as described in the manufacturer's protocol. Dialyze purified antibody in an appropriate dialysis membrane (pore size < 50-100 kDa) against 1 L of 0.15 M sodium chloride for at least 2 h or overnight.
      1. Transfer the antibody to 1 L of 0.1 M NaHCO₃ and dialyze for at least 2 h.
      2. Dissolve the required amount of fluorescent dye in dimethyl sulfoxide (DMSO) (10.8 µL/mg of antibody) and add it to the antibody solution. Use the fluorescent dye in 20-fold molar excess to achieve a dye-to-protein ratio of 1:3.
      3. Incubate in the dark at 4 °C for 1 h. Remove unlabeled antibody by dialysis against 1 L of 0.15 M sodium or by using a PD-10 desalting column and resolve in phosphate-buffered saline (PBS) for in vivo application.
      4. Determine the final antibody concentration and labeling ratio by spectrophotometry.
      5. Measure the protein concentration at an absorption of 250-330 nm and consider additional absorption by the dye. Correct the maximum absorption at 280 nm (protein) by 11% of the maximum absorption at 750 nm (next step) for Cyanine7 labeling.
      6. Measure a dilution of the compound (typically 1:10) at 250-800 nm and extract the concentration of Cyanine7 at 750 nm.
      7. Determine the dye-to-protein ratio as: dye/antibody = maximum absorption at 750 nm / 200,000 / (maximum absorption at 280 nm - 0.11 x max absorption at 750 nm) / 170,000.
      8. Keep the antibody solution at 4 °C and shield it from light to avoid bleaching before injection.
   2. Load the necessary antibody solution volume in a sterile syringe immediately before injection and shield from light until it is used.
   3. Determine the optimal timing of probe injection and the scanning procedure, depending on tracer pharmacokinetics.
   4. Anesthetize the mice using 1.5 - 2.5% inhaled isoflurane in oxygen or place them securely in a dedicated restrainer for the tail vein injection of the labeled antibodies .
   5. For full-length antibodies, inject the labeled antibody at least 24 h prior to scanning, such as for anti-mouse F4/80 macrophage visualization in murine colitis. Inject mice intravenously (i.v.) via the tail vein with labeled antibody in an amount corresponding to 2.0 nmol of dye .
   6. Use equally labeled unspecific antibodies (e.g., rat IgG or another isotype corresponding to the primary antibody heritage) as an isotype control in doses equivalent to those of the specific probe.
      NOTE: The results of in vivo scans after injection of the control compound can serve as a reference for the interpretation of the specific probe imaging data.
   7. Use an electric razor to shave the animal fur in the abdominal region to minimize light reflection and absorption.

2. Technical Equipment

1. Use a veterinary fluorescence-mediated tomography (FMT) device for small-animal fluorescence ( Figure 4).
2. Create a new study for each project by clicking the "new study" button and include in the study description the relevant tracers, including the imaging parameters and doses for future reference.
3. Within this study, create study groups according to the respective study design (e.g., for the specific tracer and unspecific isotype control) by clicking the "new study group" button. Equip each study group with the respective number of animals.
4. Calibrate the system for the tracer constructs.
   1. Perform the calibration for each batch of tracers to normalize for the variation in labeling and enable quantitative measurement from OI data.
   2. Follow the instrument manufacturer's guidance for the calibration of each individual system; upon selection of "add new tracer," the system will provide a guide through the steps. Provide the dilution of the applied antibody solution and the calculated absolute concentration of tracer in the probe.
   3. For FMT, use a tissue-mimicking phantom of defined thickness and absorption characteristics (resembling vital tissue) and fill it with a specific volume of the antibody solution used. Measure this on the FMT device.
      NOTE: The system will use the reference measurement of the provided probe, together with the given concentration, to calculate absolute tracer concentrations from future in vivo measurements.
5. Use a heatable examination cassette with a temperature of 42 °C.
   NOTE: This prevents the mice from becoming hypothermic during the examination.

3. Animal Anesthesia

1. Use a continuous flow of 1.5 - 2% vol% isoflurane ([2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane]) and 1.5 L O₂/min to anesthetize the mice. Use specially designed inhalation systems for rodent anesthesia (isoflurane vaporizer) to easily control the anesthetic depth and to minimize staff exposure.
2. Place the mouse in the leak-proof induction chamber and turn on the vaporizer for isoflurane supply (100% (v/v), 5 vol% in oxygen, 3 L/min). Monitor the mouse until it is recumbent and unconscious.
3. Place it in the examination cassette for tomography, with continuous isoflurane inhalation via nose cone at a dose of 100% v/v, 1.5 vol% in oxygen, 1.5 L/min to minimize movement artefacts during examination. For procedures lasting longer than 5 min, apply eye ointment to the mouse eyes to prevent corneal damage.
4. Assess anesthetic depth by checking the reflexes. Lay the mouse on its back; if anesthesia is sufficient, the mouse should not turn around. Pinch the mouse softly between its toes; if anesthesia is sufficient, the leg will not be withdrawn (stage of surgical tolerance).

4. Fluorescence-mediated Tomography Scan

NOTE: Adapt the following details, which are specific to the FMT system used in this study (see the Table of Materials) for alternative fluorescence reflectance imaging devices or FMT systems, as needed.

1. Place the anesthetized mouse on its back in the examination cassette.
2. Perform the scanning procedure.
   1. Insert the cassette into the imaging system and close it immediately to ensure continuous anesthesia. Select the appropriate sample from the study group created previously. Select the administered tracer from the dropdown menu to ensure that the values for tracer concentration are properly calculated.
   2. Acquire fluorescence reflectance image at the appropriate wavelength (720 nm for Cyanine7) for scan planning and outline the scan field by clicking the "acquire image" button.
   3. See that the scan field appears as an overlay on the fluorescence reflectance image. Adjust it to the region of interest (e.g., colon or abdomen), avoiding air or areas of remaining fur. Depending on the image target, set the number of image data points within the scan field by choosing from the coarse to medium and fine scan-field resolution in the menu on the right.
      NOTE: Keep in mind that a fine scan field might offer better spatial resolution at the cost of a significantly longer scanning time.
   4. Start data/image acquisition at the selected wavelength by clicking "scan."
      NOTE: The scan time for a medium-fine scan of the whole abdomen will be around 5 min; in this time, each data point is separately illuminated by the excitation laser, and the resulting fluorescence is recorded.
   5. Remove the animal from the imaging cassette at the end of the scan and allow the animal to recover entirely before placing it back into the cage.
   6. Repeat the FMT if deemed necessary at various time points during the experiment (e.g., days 0, 5, and 9 - 10 (end of the experiment)), but consider the accumulation of antibody in the body, therefore increasing the background fluorescence signal.

5. Post-scan

1. Place the mouse in a separate cage on a paper towel under a red-light warming lamp and monitor the mouse for signs of discomfort or distress until full recovery. Place the mouse back into its respective cage when fully awake.
2. Euthanize the mice at the end of the experiment by the delivery of CO₂ to the isolator at a dose of 100% (v/v), 100 vol%, and 3 L/min. Do not pre-fill the chamber with CO_2, as a sudden exposure to high concentrations of CO₂ might cause distress. Verify death after the mouse has stopped breathing by a subsequent secondary mode of euthanasia such as rapid cervical dislocation.
3. Explant the colon by abdominal laparotomy and perform an ex vivo scan of the explanted colon, as explained in steps 4.2.1 - 4.2.4. Open each colon longitudinally using Metzenbaum surgical scissors and thoroughly rinse with saline solution before preparing it for further analysis.
4. Use a scalpel to cut a 0.5-cm fragment from the distal colon and immediately place it in a 1.5-mL cryogenic tube. Freeze it in liquid nitrogen and store at -70 °C until further use (e.g.,myeloperoxidase (MPO) measurements).
5. Use a wooden stick and roll up the remaining longitudinally opened colon from the distal to proximal end, with the mucosa outwards ("Swiss roll technique"), for histological analyses. Place the preparation in a fixative (see the Table of Materials) and freeze at -80 °C¹⁴.

6. Data Reconstruction and Interpretation

1. Use the reconstruction tool of the respective imaging software to create 3D maps of the fluorescence distribution from the raw imaging data; scans are automatically added to the reconstruction tool, when upon scanning, the function "add to reconstruction queue" is selected.
   1. Otherwise, select the scans from the dropdown menu under the respective study and study group, right-click the scan, and select "add to reconstruction."
2. For further analysis, load a data set into the analysis software. From the dropdown menu in the top bar, select the respective study and then select the study group and the individual animal from the menu on the right; all scans performed for this animal will be shown. Select the correct scan and click "load."
   NOTE: The 3D reconstruction of the tracer distribution will appear on the left as an overlay of the initially acquired fluorescence reflectance image. The model can be rotated and magnified for easier analysis.
3. Identify foci of unspecific label accumulation (e.g., liver or urine bladder) on reconstructed 3D maps and differentiate from target tissues (e.g., bowel or intestine).
4. From the top bar, select the ROI shape most appropriate for the target. Label target tissue as regions of interest (ROI) by placing the respective measuring tools in the analysis software; the software will provide a fluorescence intensity for the ROI, as well as (pico-)molar amounts of the tracer that the scan has been calibrated for.
5. Select the total amount of tracer in the appropriate ROI, normalized for the ROI size, as the most suitable equivalent for the evaluation of disease activity in correlation with the inflammatory infiltrate (histology).
   NOTE: Other features of the ROI can be chosen if appropriate as representative of the particular model.

7. Ex Vivo Analyses

1. Hematoxylin and eosin staining and immunofluorescence staining.
   1. Deparaffinize sections by placing them in 70% ethanol (EtOH) for 2 min; rinse with distilled water afterwards. Stain with hematoxylin solution for 5 min and subsequently rinse with warm tap water for 10 min.
   2. Rinse with distilled water, counterstain with eosin solution for 2 min, and again rinse with distilled water.
   3. Place in 70% EtOH, 96% EtOH, 99% EtOH, and xylene (2 times) for 2 min each to dehydrate and clear the sections. Mount with resinous mounting medium.
2. Immunofluorescence staining.
   1. Use the previously obtained tissue sections ("Swiss roll") for immunofluorescence staining and prepare cryo-cut sections of 7 µm.
   2. Block acetone-fixed and frozen colon sections in 5.0% rat serum for 10 min and incubate overnight with diluted (1/500 v/v) biotinylated primary rat anti-mouse F4/80 antibody. Wash the sections thrice in Tris-buffered saline (TBS) and incubate with Streptavidin-FITC (1:100 v/v) in PBS/BSA (0.1% w/v) overnight at 4 °C.
   3. Wash the sections in TBS and stain with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000 v/v) to achieve contrast. Analyze the fluorescence images under a confocal microscope (see the Table of Materials; 40x magnification; filter cube N2.1 with excitation filter 515 - 560) and count the F4/80-positive cells per high-power field.
      NOTE: Consider using antibodies of the same clone and format when combining in vivo FMT and post mortem histology analyses such as immunohistochemistry or immunofluorescence staining, depending on the intended target. For the detection of F4/80-positive macrophages in vivo and ex vivo, different formats were used.
3. MPO measurements in colon samples.
   1. Use freshly obtained, PBS-rinsed colon samples or thawed specimen for MPO measurements. Weigh all samples and homogenize the tissue in lysis buffer provided in the ELISA kit (see the Table of Materials) at a volume of 20 µL of lysis buffer per mg of tissue.
   2. Sonicate for 15 s (sonication frequency: 20 kHz, power: 70 W) and centrifuge the samples for 10 min at 200 x g and 4 °C.
   3. Use a commercially available ELISA kit (see the Table of Materials) according to the manufactures description. Carry out the test in duplicates.

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

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