Using Fluorescence-Mediated Tomography to Assess Murine Intestinal Inflammation

Published: April 30, 2024

Abstract

Source: Nowacki, T. M., et al. Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation. J. Vis. Exp. (2017)

This video demonstrates a technique for assessing intestinal macrophage infiltration in a murine model of colitis using fluorescence-mediated tomography (FMT). Upon inducing colitis in a mouse via dextran sodium sulfate (DSS), the animal is injected with antibodies that recognize the F4/80 glycoprotein marker on infiltrated macrophages in the colon. The mouse is then imaged in an FMT device where the fluorescence and reflectance signals are reconstructed into a three-dimensional image to determine the spatial distribution and intensity of fluorescence.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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 body weight.
    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 mice.
      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, λexcitation: 750 nm, λemission: 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 antibodies 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 Cyanine 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 it 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 1).
  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 the 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 artifacts during the 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 antibodies in the body, therefore increasing the background fluorescence signal.

5. 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.

Representative Results

Figure 1
Figure 1. Tomographic visualization of macrophage infiltrates in murine colitis.

FMT scans in colitic mice and non-colitic controls administered Cyanine7-conjugated antibody against murine F4/80 (n = 5 per group). (A) Bar graph: total fluorescence intensity in pmol of dye was determined in the ROI and depicted ± SEM. Representative images are shown with color-coded fluorescence intensity corresponding to the extent of inflammatory infiltrate in mice injected with the specific probe (anti-mouse F4/80) (B) and an unspecific control (rat IgG) (C). In both cases, an ROI of equal size was placed transversely in the upper abdomen for further analysis.

Disclosures

The authors have nothing to disclose.

Materials

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
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

Tags

Play Video

Cite This Article
Using Fluorescence-Mediated Tomography to Assess Murine Intestinal Inflammation. J. Vis. Exp. (Pending Publication), e22112, doi: (2024).

View Video