Using Fluorescence-Mediated Tomography to Assess Murine Intestinal Inflammation

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, 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 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.