Competitive Kick boxers with rank A with a minimum of 20 competitive bouts and 3 training sessions per week were recruited from gyms in the Netherlands. Rank A is the highest rank in kickboxing allowing participation in full contact bouts and can only be achieved after several years of intensive training. Kick boxers were not allowed to have fought a recent match or sustained a recent concussion (within 3 months of the scan). Healthy, age-matched controls without a history of traumatic brain injury or participation in contact sports were recruited with local advertisements. All participants denied the use of anti-inflammatory drugs or anabolic steroids.
All subjects were asked to fill out two questionnaires: a quality of life scale (SF-36) 24, which is routinely used in the follow-up of mild traumatic brain injury by the Department of Neurology of the University Medical Center Groningen, and the Hospital Anxiety and Depression scale 25. In addition, information was gathered in relation to sociodemography, medication, use of alcohol and cigarettes, disease history, history of mild traumatic brain injury and fighting record (wins-losses-draws). All subjects underwent neuropsychological testing, with tests for the following domains: intelligence (Dutch version of the National Adult Reading Test 26), mental speed and attention (Trail Making Test (TMT) A and TMT B 27, Vienna Test System (VTS) 28, the 15 Words Test 29 (both Immediate and Delayed Recall (IR, DR)), digit span test 30, executive functioning (Zoo Map, subtest of the Behavioural Assessmentof the Dysexecutive syndrome (BADS 31)), Hayling test 32, Controlled Oral Word Association Test (COWAT, 33), and emotion recognition (FEEST) 34). Laboratory testing included serum C-reactive protein (CRP) and interleukine-6 (IL-6) as peripheral markers of inflammation.
[11C]-PK11195 was labeled by trapping 11C-methyl iodide in a solution of 1 mg of (R)-N-desmethyl-PK11195 and 10 mg of potassium hydroxide in 300 μL of dimethylsulfoxide. The reaction mixture was allowed to react for 1 min at 40°C, neutralized with 1 mol/L HCl, and passed through a 45-μm Millex-HV filter (Millipore). The filtrate was purified by high-performance liquid chromatography (HPLC) using a μBondapak C18 column (7.8 × 300 mm) (Waters) with acetonitrile/25 mM NaH2PO4 (pH 3.5; 55/45) as the eluent (flow, 5 mL/min). To remove the organic solvents from the product, the collected HPLC fraction (retention time, 7 min) was diluted with 100 mL of water and passed through an Oasis HLB 30-mg (1 mL) cartridge. The cartridge was washed twice with 10 mL of water and subsequently eluted with 1 mL of ethanol and 8 mL of water. The product was sterilized by filtration over a 0.20-μm Millex-LG filter (Millipore). Quality control was performed by HPLC, using a Novapak C18 column (150 × 3.9 mm) (Waters) with acetonitrile/25 mM NaH2PO4 (pH 3.5) (60/40) as the eluent at a flow of 1 mL/min. The radiochemical purity was always greater than 95%. No differences were found between the net injected dose in healthy volunteers (333 ± 53 MBq) and that in patients (336 ± 27 MBq) (p = 0.679).
Subjects fasted for 6 hours prior to the PET scan. [11C]-PK11195 was injected with a speed of 0.5 mL/second followed by a 60 minute dynamic PET scan consisting of 23 frames with increasing length on a Siemens Biograph mCT system (Siemens Medical Solutions USA, Inc) with the head immobilized in a headrest to reduce motion artifacts. Images were reconstructed using Truex+TOF with 3 iterations and 21 subsets in a 400 x 400 matrix size (zoom 1.0). After intravenous injection of [11C]-PK11195, arterial blood radioactivity was continuously monitored with an automated sampling system. In addition, 5 extra blood samples were collected at 10, 20, 30, 45 and 60 min after [11C]-PK11195 injection to determine the amount of radioactivity in blood and plasma for calibration of the blood monitor, and to determine the amount of radioactive metabolites in plasma. MRI-T1w images were acquired for PET co-registration purposes. Subjects were scanned at a Philips Intera 3.0T MRI scanner (Philips, Best, The Netherlands) with a 32-channel head coil. A 3D T1w TFE image was acquired for each subject using the following parameters: 160 sagittal slices without gap, FOV (ap × rl × fh) 256 × 160 × 256 mm, acquired matrix 256 × 256, voxel size 1 × 1 × 1 mm, repetition time 9 ms, echo time 3.5 ms and flip angle 8 degrees. Diffusion-weighted imaging (DWI) was performed using a single-shot spin-echo echo-planar imaging sequence with the following parameters: 65 axial slices of 2 mm without gap, FOV 240 × 240 × 130 mm3, acquisition matrix 117 × 120, reconstructed voxel size 1.88 × 1.88 × 2 mm3, TR 8877 ms, TE 60 ms, flip angle 90°, 60 diffusion directions (and seven non-diffusion-weighted scans averaged to one volume), and b-value 0 and 1,000 s/mm2. The noise of the diffusion scans was first reduced 35,36 and corrected for Gibbs ringing artefacts 37. This was followed by motion correction with correction for eddy-current induced distortions 38,39and bias field correction 40. Subsequently, the diffusion data were subjected to diffusion tensor analysis using FMRIB Software Library (FSL v6.0.4.) 41.
All PET images were coregistered to the individual anatomical T1w MRI and spatially normalized to Montreal Neurological Institute (MNI) space using PMOD (version 4.0, PMOD Technologies Ltd, Zürich, Switzerland). Brain regions were defined using the Hammers maximum probability atlas (Hammers N3083) containing frontal lobes, parietal lobes, temporal lobes (with exception of the amygdala and the hippocampus, which were analyzed separately), occipital lobes, cerebellum, striatum and white matter. In addition a whole brain grey matter (GM) region was generated, combining all cortical regions. For each patient, the binding potential (BPND) was calculated, defined as k3/k4, and used as outcome measure, using a 2 tissue compartment model with K1/k2 fixed to the value obtained for whole brain GM . For kinetic modeling, frame length dependent weighting was used for fitting the time activity curve, a correction for blood delay was used and blood data was fitted 42. Fixing of k4 to the whole brain GM value was performed in subregions when this parameter could not be adequately calculated (e.g. in case of a very high standard error (>25%), occurring in 7 subjects). Group level comparisons were performed using independent sample Mann-Whitney U tests, with statistical threshold for significance set at 0.05. A non-parametric test was chosen because it was anticipated that a non-normal distribution could be present in kickboxers with individual outliers in C-PK11195 binding depending on the RPI exposure. For statistical analysis, SPSS was used (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp). Correlations between regional BPND and the scores on the neuropsychological testing and questionnaires were assessed using scatter plots and linear regression analysis using GraphPad Prism version 7.02 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).
Diffusion tensor imaging data were generated with FSL. The fractional anisotropy (FA) and mean diffusivity (MD) maps were spatially normalized to the ICBM maximum probability maps using PMOD. Atlas based white matter regions were automatically defined . FA and MD values of predefined white matter regions (Figure 1, corpus callosum (genu, body, splenium), anterior limb of internal capsule, posterior limb of internal capsule, retrolenticular part of the internal capsule, anterior corona radiata, superior corona radiata, posterior corona radiata, posterior thalamic radiation, superior longitudinal fasciculus) were measured. Similar to previous DTI studies in sports related repetitive head injury, these white matter regions were selected in order to be sensitive to potential changes related to projections from widespread areas of the brain, especially long axonal projections 22. Additional MRI sequences included SWI, T2w flair and DTI. All MRI images were visually assessed by a neuroradiologist (AvdH) for the presence of structural abnormalities.