General methods
Radioactivity was counted using a CRC-R dose calibrator (Capintec, Inc., Ramsey, NJ, USA), while low-level counting (< 60 kcps) was done using a Caprac-R well counter (Capintec, Inc.). An Inveon dedicated PET scanner (Siemens Medical Solutions, Inc., Malvern, PA, USA), which has a resolution of 1.46 mm in the center of the field of view [21], was used for the PET studies. An Inveon Multimodality [MM] CT scanner (Siemens Medical Solutions, Inc.) was used for CT acquisitions in combined PET/CT experiments. All in vivo and ex vivo images were analyzed using ASIPro VM (Siemens Medical Solutions, Inc.), PMOD Software (PMOD Technologies Ltd., Zurich, Switzerland), and Inveon Research Workplace [IRW] software (Siemens Medical Solutions, Inc.). Slices of BAT were prepared using the CM1850 cryotome (Leica Microsystems Inc., Buffalo Grove, IL, USA). Ex vivo18F-FDG-labeled sections were exposed to phosphor films and read using the Cyclone Phosphor Imaging System (Packard Instruments, Meriden, CT, USA) and were analyzed using the OptiQuant software (Packard Instruments). All animal studies were approved by the Institutional Animal Health Care and Use Committee of the University of California-Irvine.
Animals
Male Sprague-Dawley rats, aged 11 to 12 weeks, weighing 343 ± 9 g at the beginning of the experiment, were used in this study. The rats were purchased from Harlan Laboratories, Inc. (Placentia, CA, USA) and housed under controlled temperatures of 22°C ± 1°C in a 12-h light-dark cycle, on at 6 a.m., with water and food chow ad libitum.
Experimental protocol
General procedures
All rats were fasted for 24 h before 18F-FDG administration. The control, cold-exposed, and CL316,243- (Tocris Bioscience, Ellisville, MO, USA) and propranolol-pretreated (Sigma-Aldrich Corporation, St. Louis, MO, USA) rats were administered intravenously [i.v.] with 14 ± 1.5 MBq 18F-FDG under 2% isoflurane anesthesia. Following the injections, the rats were awake for 60 min and subsequently placed in the supine position in a rat holder and anesthetized with 2% isoflurane for upper-body PET imaging. The rat holder was placed on the PET/CT bed, and all animals had a CT scan after the PET scan for attenuation correction and anatomical delineation of PET images.
Drug effects
The control and CL316,243-pretreated rats (CL316,243, 2 mg/kg i.v., 30 min before 18F-FDG administration) were anesthetized for upper-body PET imaging (n = 3, each group). The same method of injection of 18F-FDG was used for all rats, as was the recovery period and re-anesthetization for PET. Dose effects of CL316,243 were investigated by injecting 0.1, 0.5, and 1 mg/kg of CL316,243 30 min prior to 18F-FDG administration. To evaluate whether enhanced 18F-FDG uptake in activated BAT could be reduced by pharmacologic interventions, 5 mg/kg propranolol was given intraperitoneally in the anesthetized rats 30 min prior to CL316,243 administration. Immediately after PET imaging, the rats were sacrificed; BAT and white adipose tissue [WAT] were harvested for autoradiography and counted in the well counter.
Temperature effects
Cold-exposed rats (n = 3, each group; cold treatment was at 8°C for 120 min prior to PET imaging) were administered i.v. with 18F-FDG under 2% isoflurane anesthesia. During cold exposure, the rats were caged alone without any sand bedding. Following the injections, the rats were awake for 60 min at ambient temperature (for control) and 8°C (for cold treatment) and subsequently anesthetized with 2% isoflurane for PET imaging.
18F-FDG PET/CT imaging
The Inveon PET and MM CT scanners were placed in the 'docked mode' for combined PET/CT experiments. After 60 min of 18F-FDG uptake, the rats were anesthetized and placed in supine position with their chest, neck, and head within the field of view of the PET scanner. PET data were acquired for 30 min, followed by a CT scan (large area detector, 10 cm × 10 cm field of view) for attenuation correction and anatomical delineation of PET images. The CT projections were acquired with the detector-source assembly rotating over 360° and 720 rotation steps. A projection bin factor of 4 was used in order to increase the signal-to-noise ratio in the images. The CT images were reconstructed using cone-beam reconstruction with a Shepp filter with a cutoff at the Nyquist frequency and a binning factor of 2, resulting in an image matrix of 480 × 480 × 632 and a voxel size of 0.206 mm. The PET images were spatially transformed to match the reconstructed CT images. PET images were corrected for randoms, attenuation, and scatter and were reconstructed as 128 × 128 × 159 matrices with a transaxial pixel of 0.776 mm and a slice thickness of 0.796 mm using a fast maximum a posteriori [fastMAP] algorithm in conjunction with three-dimensional ordered subset expectation maximization [OSEM3D] (2 OSEM3D iterations, MAP 18 iterations, 0.1 smoothing factor). All images were calibrated in units of becquerels per cubic centimeter by scanning a 68Ge cylinder (diameter 6 cm) with a known activity and reconstructing the acquired image with parameters identical to those of 18F-FDG images.
Image analysis
18F-FDG quantitation
The magnitude of BAT 18F-FDG activation was expressed as the standard uptake value [SUV] which was defined as the average 18F-FDG activity in each volume of interest [VOI] (in kilobecquerels per cubic centimeter) divided by the injected dose (in megabecquerels) times the body weight of each animal (in kilograms). The SUVs were thus expressed in units of [kilobecquerels per cubic centimeter per (megabecquerel per kilogram)].
Volume measurement of BAT
The interscapular, cervical, periaortic, and intercostal BAT were visualized on 3-D CT images with the 3-D Visualization toolbox of the IRW software. For quantitative analysis, VOIs were drawn on PET images using PMOD. Similar to as described previously [22], the VOIs were first delineated visually by contouring the 18F-FDG activity that was clearly above the normal background activity (Figure 3).
In order to measure the volume of activated BAT, a new VOI contour was delineated based on a threshold equal to the mean 18F-FDG activity minus one standard deviation of all voxels within the primary visually defined VOI. The volume of the newly delineated VOI was used to report the activated BAT volume.
Autoradiography
Transverse sections obtained from the soft tissue of the interscapular BAT [IBAT] region at the level of T5 were frozen on dry ice to provide 60-μm-thick slides for autoradiography. The slides were exposed to phosphor screens for 1 h and scanned using the Cyclone Plus storage phosphor system. The acquired image was then analyzed with the OptiQuant™ software where data in each ROI were quantified in digital light units per millimeter squared [DLU/mm2].
To confirm IBAT activation, biopsies were taken for histological evaluation (hematoxylin and eosin [H&E] staining) using 20-μm-thick frozen sections. Samples were viewed by light microscopy to determine the presence of multilocular droplets. Images were saved as TIFF files (16 bits) and were quantified using the ImageJ software for light intensity (range 0 to 256).
Statistical analysis
Statistical differences between groups were determined using either independent Student's t test or one-way ANOVA with a Bonferroni post hoc test in the SPSS statistical software, version 16.0 for Windows (Chicago, IL, USA). A p value of < 0.05 was considered to indicate statistical significance.