Arterial spin labeling versus BOLD in direct challenge and drug-task interaction pharmacological fMRI

Study participants

Fourteen nondemented, nondepressed, ambulatory adults age 40–75, 11 men, with idiopathic Parkinson disease, Hoehn & Yahr stage 1–3 (mean stage 2) (Hoehn & Yahr, 1967), treated with a stable dose of levodopa but no dopamine agonists, participated in the clinical trial (registered at http://clinicaltrials.gov with identifier NCT00605553). Detailed inclusion and exclusion criteria were reported previously (Black et al., 2010a). The study was approved by the Washington University Human Research Protection Office (IRB), approval # 08-0059, and all subjects provided written documentation of informed consent prior to participation.

Study protocol

Subjects were randomly assigned to one of two treatment groups: one group took 60 mg of the adenosine A2a antagonist tozadenant (SYN115) twice daily for one week, waited for a one week washout period and then took a matching placebo twice daily for one week; those assigned to the other group participated in the reverse order. The original report included additional subjects allocated to 20 mg vs placebo, but for this report we focus only on the 60 mg arms. Adenosine A_2a antagonists have been studied eagerly as potential treatments for Parkinson disease, alone or in combination with standard antiparkinsonian therapy (Pinna, 2014). A_2a receptors occur together with dopamine D₂ and D₃ receptors on striatopallidal neurons that inhibit the indirect basal ganglia pathway, and A_2a antagonists mimic some of the biological effects of dopamine D₂ and D₃ agonists (reviewed in Black et al., 2010b).

Subjects and investigators were blind to the group assignments. Neuroimaging was performed on the last day of each treatment week. On the morning of the scan day, they did not take their usual antiparkinsonian medications, but did take the last dose of tozadenant or placebo at approximately 6:00 AM. The timing of the fMRI assessments was planned to approximately bracket the time to maximal plasma concentration of tozadenant after chronic dosing. Subjects took 200 mg of carbidopa on arrival to the imaging center and then underwent two sets of MRI assessments, once before and once during an infusion of levodopa, dosed to produce a steady plasma concentration of 600 ng/mL. Levodopa is a precursor to dopamine and is used in Parkinson’s disease to ameliorate the deficiency of dopamine in the substantia nigra. The carbidopa pretreatment was given to inhibit peripheral metabolism of the upcoming levodopa infusion, minimizing side effects from dopamine production in the periphery and keeping more of the levodopa available to the brain.

Subject behavior

Each scanning session included two perfusion MRI (ASL) runs while the subject performed the 2-back memory task, two control ASL runs while the subject fixated on a crosshair, and two block-design BOLD runs, each with four fixation blocks bracketing three task blocks (Fig. 1). ASL scans were also obtained for additional tasks without a BOLD comparison. In each session the fixation ASL and 2-back ASL scans were acquired immediately after the BOLD runs. The 2-back task inter-stimulus interval was 2.5 s for both ASL and BOLD.

This study employed a working memory task for several reasons. Working memory performance is affected by Parkinson disease and is sensitive to manipulations of central dopaminergic transmission (Cools & D’Esposito, 2011; Hershey et al., 2004). Adenosine A_2a receptor antagonists interact with dopamine receptors and can improve working memory performance (Takahashi, Pamplona & Prediger, 2008), including in animal models of parkinsonism (Kadowaki Horita et al., 2013). Several cognitive-pharmacological interaction phMRI studies have employed working memory tasks (Barch et al., 2012), including another study with tozadenant (Moeller et al., 2012). For these and other reasons, several A_2a antagonists have been studied for possible cognitive benefits in PD (Pinna, 2014).

One subject was excluded from all analyses presented here because his 2-back task performance was less than 70% accurate. All other subjects had greater than 70% on every run. We previously reported that tozadenant at this dose had no statistically significant effect on 2-back performance, including accuracy and response time (Campbell et al., 2010).

MR image acquisition

All MRI data were acquired at 3T on the Siemens Magnetron Tim Trio with the 12-channel matrix head coil. BOLD-sensitive echo-planar images (EPI) were obtained with flip angle 90°, echo time (TE) 27 ms, repetition time (TR) 2000 ms, 36 planes with interleaved slice acquisition, field of view (256 mm)², and voxel size (4.0 mm)³. Over a period of 4.33 min for each run, 130 volumes (frames) were acquired; the first 4 frames were discarded to ensure steady-state magnetization.

ASL images were acquired with the commercial Siemens pulsed ASL (pASL) sequence (Wang et al., 2003b). Fifteen transverse echo-planar readout slices with center-to-center slice distance 7.5 mm were acquired with (64)² (3.4375 mm)² voxels in each plane, TE 13.0 ms, TR 2600 ms, and flip angle 90°. Labeling slab thickness was 10 cm. Fat suppression was used. The perfusion mode was PICORE Q2T, with TI₁ 700 ms, saturation stop time 1600 ms and TI₂ 1800 ms. An M₀ image was followed by 31 tag–control pairs for a total acquisition time for each ASL run of 2.73 min.

Brain structure was assessed from sagittal magnetization-prepared rapid gradient-echo (MP RAGE) acquisitions with voxel size (1.0 mm)³, TE = 3.08 ms, TR = 2400 ms, TI = 1,000 ms, flip angle = 8° (Mugler III & Brookeman, 1990), one at each of the 4 scanning sessions. The structural images for each subject were inspected visually, images of lower quality were rejected, and the remaining 1-4 MP-RAGE images for each subject were mutually registered and averaged using a validated method (Black et al., 2001).

Image preprocessing

BOLD images from each subject were preprocessed to reduce artifacts, including correction for intensity differences due to interleaved acquisition, interpolation for slice time correction, correction for head movement, and alignment to atlas space (Hershey et al., 2004). Image intensity was adjusted on a frame-by-frame basis so that each frame had a whole-brain modal value of 1,000 (Ojemann et al., 1997). Frames were smoothed using a 6 mm (FWHM) Gaussian filter and resampled to (3 mm)³ cubic voxels. To minimize motion-related artifact, frames were removed if framewise displacement exceeded 0.9 mm (Siegel et al., 2014).

The 63 frames of the ASL run were smoothed using a 5.7 mm (FWHM) Gaussian filter (resolution chosen to best match the final smoothing estimated from the BOLD images) and rigidly aligned using a method validated in humans and other species (Black et al., 2001; Black et al., 2014). Cerebral blood flow (CBF) was computed in each voxel for each tag-control EPI pair as described (Wang et al., 2003b). The aligned EPI images were also summed to facilitate later alignment steps, and the summed, aligned EPI images from each run were mutually aligned within each subject and summed across runs. The resulting summed EPI images from each subject were affine registered to a target image in Talairach and Tournoux space made using validated methods from these subjects’ structural MR images (Hershey et al., 2004). The products of the registration matrix from this step and the matrices from the within-run mutual registration step were used to resample the 31 tag–control pair CBF images from each run into atlas space images with (3 mm)³ cubic voxels in a single resampling step. As with the BOLD, to minimize motion-related artifact we removed tag–control pairs if framewise displacement in either EPI image exceeded 0.9 mm (Siegel et al., 2014). One subject’s data was excluded from further analysis because over half of his frame pairs were removed due to head motion, leaving 12 subjects for all analyses below. The CBF images in atlas space from the remaining pairs were averaged to create one atlas-registered CBF image for each ASL run. Each CBF image was scaled to a modal global (whole-brain) CBF of 50 mL/hg/min (Stewart et al., 2014). See Fig. 2 for an example CBF image.

Statistical analysis

Secondary analysis: effects of levodopa

The study design was optimized for tozadenant rather than levodopa, and the levodopa dose was relatively low, so analyses examining the effect of levodopa were secondary. To investigate the effects of levodopa we created statistical images of the levodopa effect (comparing scans acquired during the levodopa infusion to scans prior to infusion), of the interaction of the 2-back task with levodopa, and of the 3-way interaction of the 2-back task, levodopa and tozadenant.

Cross-modality image comparison

The final resolution of the (3 mm)³ ASL and BOLD images was similar (Table 1). Total acquisition time was about 25% longer for ASL than BOLD, but acquisition time for the data actually submitted to statistical analysis was much more similar (Table 1), largely because each head movement lost 5.2 s of data in the ASL data versus 2.0 s in the BOLD data.

Task activation

The working memory task serves as a positive control, and significant regional activations were identified. The analysis using the ASL data identified one significant activation cluster (22 voxels = 0.6 ml, corrected p = 0.030, peak t = 5.88 at −32, −3, 57, left middle frontal gyrus, Brodmann area [BA] 6) (Fig. S1). The analysis using the BOLD data identified 12 significant clusters; the largest cluster also included −32, −3, 57 (515 voxels = 13.9 ml), corrected p < .001, peak t = 12.29 at −40, 3, 33 (left precentral gyrus, BA6) (see Table 2, Fig. S2A). There were no significant deactivations in the ASL data, while the analysis using the BOLD data identified 11 significant deactivation clusters (the largest had volume 2,142 voxels = 57.8 ml, corrected p < .001, peak t = 12.70 at −4, −54, 12, left posterior cingulate, BA29) (Table 3, Fig. S2B).

Drug effect

The task-drug interaction (tozadenant ×2-back) showed no significant results for ASL or BOLD (Figs. S3 and S4). However, the drug vs. placebo contrast from the same ASL data revealed significant rCBF decreases on tozadenant in the thalamus bilaterally (Table 4, Fig. 3, Fig. S5). There were no significant clusters of increased rCBF. As expected, the same contrast with the BOLD data found no significant clusters of activation or deactivation (Fig. S6). Table 5 summarizes all these contrasts.

Levodopa effect

There were no significant clusters for the pure levodopa effect (Figs. S7 and S8), the task- levodopa interaction (Figs. S9 and S10), or the 3-way interaction (Figs. S11 and S12) in either the ASL or the BOLD images.