PET/MR imaging allows for both qualitative and quantitative measurements on these metabolically-active brown and beige adipocytes in live animals so that the activity of these so-called thermogenic adipocytes can be measured on a functional basis. The main advantage of this technique is that it allows acquisition and combination of PET and MRI scans on the same subject, thus enabling precise and simultaneous acquisition of thermogenic adipocytes in different depots of the small animal. This method can be easily transferred into any preclinical system or modified into a high throughput format by simultaneously imaging of multiple mice, thereby increasing the statistical power of the imaging data at a reduced cost and time.
To harvest the interscapular brown adipose tissue, place an anesthetized 8-week-old C57 Black 6 mouse onto a heating pad and make a 2 centimeter incision along the dorsal midline. Remove the iBAT pads from the interscapular region. After the bleeding stops, use 7 millimeter stainless steel wound clips to close the incision.
When all of the iBAT has been collected, house the mice in an intensive care unit for 14 days. Administer 5 milligram per kilogram Meloxicam to the mice for six days and remove the clips as soon as the wound has healed. To enable automated sequential PET/MR scans prior to imaging session, set the discrimination level to 400 to 600 kiloelectron-volts, the confidence mode to 1-3, the scanning time to 20 minutes, and the study isotope to F-18 to set the workflow for PET acquisition.
To acquire T1-weighted magnetic resonance for attenuation correction, set the Gradient Echo-3D parameters. To acquire T2-weighted magnetic resonance as an anatomical reference, set the Fast-spin Echo 2D parameters. To allow reconstruction of the PET images, use the Tera-Tomo 3D algorithm with the coincidence mode set to 1-3 and with the decay, deadtime, random, attenuation, and scatter corrections set to create images with an overall voxel size of 0.3 cubic millimeters.
One day before the start of the imaging study to check the accuracy of the PET quantitation, put on the appropriate PPE and fill a 5 milliliter syringe with F18-FDG as recommended by the manufacturer. Use a dose calibrator to record the activity of the syringe and note the time of the measurement. In the software, use the Interpolated Ellipse ROI to draw a volume-of-interest on the reconstructed image of the syringe and compare the recovered activity to the value measured using the dose calibrator.
On the day of the imaging experiment, use forceps to carefully place the 18F-FDG stock vial behind an L-block tabletop shield and dilute an aliquot of radioisotope in 100 to 150 microliters of sterile saline to a total activity concentration of 200 to 250 microcuries. Draw the 18F-FDG solution into a 1 milliliter syringe equipped with a needle and measure the radioactivity with the dose calibrator as demonstrated. Record the weight of the mouse to be imaged before injecting the 18F-FDG solution into a tail vein.
Take note of the injection time and the residual radioactivity of the syringe to enable decay correction. Then place the mouse back into its cage for 60 minutes. To calculate the injected 18F-FDG activity, subtract the activity in the syringe before the injection from the activity measured after the injection.
At the end of the radioisotope uptake period, turn on the air heater for the mouse bed of the micro-PET/MR scanner. Place the anesthetized FDG-injected mouse onto a respiratory pad in the scanner bed and perform a scout view to determine the mouse position. Adjust the position of the mouse bed such that the entire body can be observed, ensuring that the center of the MRI field of view coincides with the center of the body.
Under PET Acquisition, select Scan Range on Previous Acquisition to use the scout view position and click Prepare to move the animal bed from MR to PET. Select OK to initiate the PET scan. Record the injection dose and time measured before and after 18F-FDG administration in the Radiopharmaceutical Editor and enter the weight of the mouse under the Subject Information menu.
Once the PET scan is complete, select Prepare to move to MR imaging and complete all MR acquisitions in the study list window. Select OK to start the MR scans. After the whole workflow has completed, briefly evaluate the quality of the acquired MR images in the post-processing software and click Home to move the mouse bed from MR to the original position.
When all mice have been imaged, to reconstruct the data in the Raw Scan menu, select PET Acquisition to load the completed PET scan and select T1-weighted MR Acquisition to allow a material map to be created. Then use the Tera-Tomo 3D algorithm to reconstruct the data as demonstrated. The removal of iBAT prior to cold treatment alters the activity of the iWAT, as indicated by the remarkable increase in 18F-FDG uptake observed in iWAT by micro-PET/MR imaging.
In addition, multilocular adipocytes, which are characteristically observed in beige adipose tissue, are more pronounced in iWAT from mice after iBAT removal compared to the adipocyte morphology observed in iWAT from sham-operated animals. Cold-treated, sham-operated mice demonstrate a markedly elevated 18F-FDG uptake in iBAT. Mice with their iBAT removed prior to cold treatment show the highest 18F-FDG uptake in the iWAT.
Indeed, cold exposure causes a sevenfold increase in 18F-FDG uptake in the iBAT of sham-operated mice, while the removal of iBAT in the cold-induced mice results in an eightfold increase in 18F-FDG uptake compared to thermo-neutral mice in which only a modest twofold increase is observed. When attempting this procedure, placing each mouse in a same position for each PET/MRI acquisition is important to improve the visualization and quantification of brown and beige adipocytes in different regions. Using this method and together with other MRI methods, such as diffusion with imaging or thermometry, more insights into the spatial distribution and prevalence of brown and beige adipocytes in mice can begin, which can potentially be translated into humans.
This method allows the researchers to study the function of these thermogenic brown and beige adipocytes. Therefore, they are especially powerful when studying the nonclinical thermogenic pathways wherein those classical markers are not altered or upregulated.
