Human Subjects
The Johns Hopkins Institutional Review Board approved this study, and all research participants provided written, informed consent. Adult (≥ 18 years of age) individuals completed a screening clinical research interview and laboratory testing (blood counts, metabolic panel, coagulation studies) with electrocardiogram and urine toxicology. Eligible participants were assessed as being in stable health with no clinical abnormality on the screening assessment and structural magnetic resonance imaging (MRI). Exclusion criteria included use of cannabis, nicotine, or other recreational substances in the past six months (assessed by self-report and urine toxicology), prescribed or over-the-counter anti-inflammatory medication within two weeks prior to the PET, acute onset or exacerbation of illness/infection in the past month, contraindication to MRI, or contraindication to PET imaging with an arterial line.
Human Brain Imaging
Synthesis of 11C-CPPC. 11C-CPPC was synthesized as described by Mathews et al [16] and met all major quality measures including radiochemical purity > 95%. Molar activity of 11C-CPPC at the time injection was 316.8 ± 138.8 GBq/µmol. The mean administered mass and radioactivity of 11C-CPPC were 1.2 ± 1.1 µg (range, 0.55–3.72 µg) and 651.4 ± 62.8 MBq (range, 542.8-736.7 MBq), respectively.
MRI Acquisition and Regions of Interest. A T1-weighted Magnetization-Prepared Rapid Gradient-Echo (MP-RAGE) sequence with 0.75 x 0.75 x 0.8 mm voxel size was acquired from each participant using a 3 Tesla MRI system (Siemens MAGNETOM Prisma, Malvern, PA, USA or Philips Achieva, Best, Netherlands). MRI data were segmented using the FreeSurfer image analysis suite (http://surfer.nmr.mgh.harvard.edu/). Ten regions of interest (ROIs) were selected: total white matter, thalamus, striatum, hippocampus, as well as cerebellar, temporal, occipital, cingulate, frontal, and parietal cortices.
PET Acquisition and Reconstruction. Before the PET, each participant underwent fitting of a thermoplastic facemask, as well as insertion of a radial arterial catheter and intravenous catheter. The thermoplastic facemask was used for head fixation during the PET to minimize head motion. An attenuation map was estimated from a 6 min transmission scan that was performed with a 137Cs point source prior to the emission scan. Each emission scan started at the time of bolus intravenous injection of 11C-CPPC, with continuous list mode data collection for 90 min on a High Resolution Research Tomograph (Siemens Healthcare, Knoxville, TN) [17]. Imaging data were reconstructed using the iterative ordinary-Poisson ordered-subset expectation-maximization algorithm (6 iteration and 16 subsets, 2 mm post-smoothing) and were corrected for decay, attenuation, random activity, and scatter [18]. The reconstructed data were binned into 30 frames (four 15 seconds, four 30 seconds, three 1 min, two 2 min, five 4 min, and twelve 5 min). The reconstructed image volume spanned 31 cm x 31 cm transaxially and 25 cm axially, with image matrix of 256 x 256 x 207 voxels (voxel size of 1.22 x 1.22 x 1.22 mm).
Plasma Acquisition. Arterial whole blood samples were collected over the course of the emission scan. Plasma was immediately isolated from each blood sample using centrifugation before plasma radioactivity was counted in a cross-calibrated gamma well-counter. The previously described modified column-switching high performance liquid chromatography (HPLC) method was used to measure the fraction of parent 11C-CPPC in plasma at 5, 10, 20, 30, 40, 60 and 90 min post-injection (p.i.) [8]. Metabolite-corrected time-activity curves (TACs) were generated by applying the parent 11C-CPPC time-activities to the total plasma TACs using linear interpolation (v3.7, PMOD Technologies Ltd, Zurich, Switzerland). Ultrafiltration (Centrifree Ultrafiltration Device, MilliporeSigma, Burlington, MA) was used to measure plasma free fraction (fP) of 11C-CPPC.
PET Data Processing. PET data pre-processing steps as well as the kinetic analyses were conducted using PMOD (v3.7, PMOD Technologies Ltd, Zurich, Switzerland). Post-reconstruction inter-frame motion correction was applied as needed by frame-by-frame matching of dynamic data to a static reference frame generated from the average of the frames corresponding to 30–60 min p.i. PET data were rigidly transformed into MR space by co-registering the mean 30–60 min PET image and each of the 30 motion-corrected PET frames to the T1-weighted MRI image.
Derivation of PET Rate Constants and Distribution Volumes. Total distribution volume, VT [19], for each ROI was derived using the metabolite-corrected arterial input function and compartmental modeling (one-tissue compartment model with 3 parameters, 1TCM; two-tissue compartment model with 4 parameters, 2TCM) or Logan graphical analysis [20]. Cerebral blood volume was set to 5% of brain volume (vB) in compartmental models. Non-linear least squares regression was used to calculate the parameters of the compartmental models and the % standard error (%SE) of each estimated parameter. In Logan graphical analysis, various selections of equilibration time, t*, were evaluated across the ROIs using the 10% max error criterion, with ultimate selection of t*=30 min.
Statistics
The compartmental model fits that were applied to the regional TACs were first assessed visually before the relative goodness of fit was assessed using the F test [21]. Data are presented as mean ± standard deviation unless otherwise noted.