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

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.