Twenty-four healthy individuals (2 females, 22 males; age range = 20-47 years) were studied. Each participant had a comprehensive screening assessment that included a clinical interview, complete physical examination with the medical history, vital signs, routine blood tests, hepatitis screen, height and weight, 12-lead electrocardiogram, urine toxicology and pregnancy test (if female). Individuals were excluded if they have metal implants, claustrophobia, prior radiation exposure over the Radioactive Drug Research Committee (RDRC) annual limit for research subjects, drug/alcohol abuse, hepatitis, abnormal laboratory values, current or past neurological illness (including a history of head injury with loss of consciousness); current and /or lifetime psychiatric disorder; diabetes, cardiac disease, abnormal ECG, and insufficient arterial patency.
The study was performed under protocols approved by the Yale Human Investigation Committee, the Yale University Radiation Safety Committee, the Yale-New Haven Hospital Radioactive Drug Research Committee, and the Yale MRI Safety Committee. Subjects were recruited from Connecticut and surrounding states by paper and web advertisements, as well as personal referrals. Written informed consent was obtained from all participants at the beginning of screening after a full explanation of study procedures was performed.
Structural MR imaging was performed on a Siemens 3-T Trio system (Siemens Medical Solutions, Malvern, Pennsylvania) with a circularly polarized head coil for purposes of excluding individuals with anatomical abnormalities and anatomically coregistering with PET scans. The dimension and voxel size of MR images were 256 × 256 × 176 voxels and 0.98 × 0.98 × 1.0 mm3, respectively.
[11C]GSK189254 was prepared as previously reported [29]. Among 24 participants, 14 participants used a high-resolution research tomograph (HRRT) (Siemens/CTI, Knoxville, TN, USA), which acquires 207 slices (1.2 mm slice separation) with a reconstructed image resolution of ~ 3 mm; 10 participants used a HR plus (HR+) scanner (Siemens/CTI, Knoxville, TN, USA), which acquires 63 slices with a reconstructed image resolution of ~6 mm. PET scans were acquired for 120 min at rest with an average radioactivity dose of 303.73 ± 175.80 MBq and injected mass of 12 ± 5 ng/kg (max 22 ng/kg). Dynamic data were binned into 33 frames (6 x 0.5 min, 3 x 1 min, 2 x 2 min, 22 x 5 min) and reconstructed with the Ordered Subset Expectation Maximization algorithm, MOLAR, including corrections for attenuation, normalization, scatter, randoms, and deadtime. Image voxel size was 1.22 x 1.22 x 1.23 mm for HRRT images and 2.06 x 2.06 x 2.42 mm for HR+ images.
A summed image (0–10 min after injection) was created from the motion-corrected PET data and registered to the subject's 3T MR image, which in turn was linearly registered to a MR template by using a 12-parameter linear transform. The cerebellum was defined by an Anatomical Automatic Labeling (AAL) template [30] delineated on MR. Then each HRRT frame image was smoothed using a Gaussian filter with a full width at half maximum (FWHM) of 5.2mm for harmonization with HR+ images.
The PET scans were acquired using i.v. bolus administration of 303 ± 176 mBq or less of high-specific activity [11C]GSK189254 using a 120 min dynamic PET scan for each injection. In the first phase of the study (5-10 min), the arterial input functions were measured with an automated blood counting system (PBS-101, Veenstra Instruments, Joure, The Netherlands) with a continuous withdrawal system where the radioactivity in whole blood was measured with a calibrated radioactivity monitor. Subsequently, individual blood samples were taken and counted at various time points. Samples were centrifuged to obtain plasma and counted, and selected samples were assayed for the presence of the parent radiotracer compound that had not been metabolized. These measurements were performed by a high-performance liquid chromatography HPLC as previous study [31], are used for the fraction of plasma radioactivity unbound to protein.
Automatic regions-of-interest (ROI) (Anatomical Automatic Labeling (AAL) for SPM2) were then applied to generate time-activity curves (TACs) in 15 gray matter ROIs (amygdala, caudate, hippocampus, hypothalamus, pallidum, putamen, cerebellum, thalamus, and cortical regions of the frontal, temporal, parietal, occipital, insula, anterior cingulate, and olfactory areas). The TACs were fitted with one- and two-tissue models (1T and 2T) over 120 minutes post injection, with the arterial input function and no metabolite correction. Regional volumes of distribution (VT) for [11C]GSK189254 were computed using the two-tissue compartment (2TC) model with the parameters k4 and K1/k2 shared across all regions (2TC shared), as in previous studies [32]. For exploring possible metabolic effects on the primary results, we also normalized VT value by plasma free fraction (fp).
All outcomes were summarized descriptively and assessed for normality prior to analysis using normal probability plots and Kolmogorov test statistics. All outcomes were approximately normal. A linear mixed model was used to model the independent and joint effects of age (continuous) and brain region (within-subject factor) on VT values and VT/fp values. Slopes were estimated within each region post-hoc and decline per decade was calculated as a percent of the fitted value at age 20. Within-subject correlations were accounted for by fitting three variance–covariance structures to the data (unstructured, compound symmetry, and heterogeneous compound symmetry) and then selecting the best-fitting structure according to the Bayesian Information Criterion (BIC) [33-34]. Similar models were used when assessing the joint effects of BMI and region. Given the exploratory nature of the study, we did not adjust p-values for multiple testing, as in previous studies [22, 35]. Analyses were conducted using SAS, version 9.4 (Cary, NC).