Multiple Sclerosis Journal - October 2017 - 1471

G Datta, IR Violante et al.
survey image was used to identify the anterior commissure (AC) and posterior commissure (PC). The
spectroscopy voxel was positioned just superior to the
lateral ventricles in the midline. The spectroscopy
voxel measured 40 mm anteroposterior × 25 mm
craniocaudal × 40 mm left-right. The voxel was
selected based on recommendations for use of MR
spectroscopy in MS patients, with modification of
length to accommodate the overall volume in our full
population.15
Proton spectra were acquired using a PRESS (Point
Resolved Spectroscopy) sequence (repetition
time = 2000 ms, echo time = 30 ms, 96 averages, 1024
data points). Magnetic field homogeneity was optimised to a linewidth of ~5 Hz over the spectroscopy
voxel using the proton signal from water. Water suppression was achieved by a chemically selective saturation, the WET (water suppression enhanced through
T1 effects) method.16
MRS analyses
LCModel software (version 6.3) was used for metabolite quantification applying the internal water reference method, accounting for different water content
in grey matter, white matter and cerebrospinal
fluid.17 LCModel analyses the magnetic resonance
spectrum as a linear combination of the basis set of
complete model spectra of metabolites from a normal population library.17 Only metabolites with
Cramér-Rao bounds <20% were considered.
Concentrations of NAA, myo-inositol, glycerophosphocholine and creatine plus phosphocreatine were
used in this study. Concentrations in millimole
(mM) units were calculated for all metabolites by
the software. Patient and MRI system factors (such
as radiofrequency receiver properties affected by
variability of gain in the amplifiers of the receiver
system, head size and position) can influence the
resonance intensity in ways the analytical algorithm
cannot account for. Our analysis used 'un-corrected'
concentrations only for the same brain and the same
scan session, when normalisation values for metabolite concentrations are identical. Otherwise, we
used internal normalisations of metabolite concentration. Creatine and phosphocreatine are present in
both neurons and glia. Their summed concentration,
referred to as total creatine concentration, shows little or no change in chronic lesions or normalappearing white matter (NAWM) in MS.18 Total
creatine concentration therefore was used as the
'internal standard' for normalisation of signals from
other metabolites from the same voxel.

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PET scanning
PET scanning (Discovery RX PET/CT scanner) was
performed with a trans-axial resolution of 5.0 mm and
a radial resolution of 5.1 mm at 1 cm from the centre
of the FOV in 3D mode. [11C]PBR28 was injected as
an intravenous bolus over approximately 20 seconds
at the start of a 90 minutes dynamic PET acquisition.
Injected activities for [11C]PBR28 ranged from 223.8
to 379.6 MBq (325.6 ± 34.6 MBq, n = 44). Injected
mass for different subjects ranged from 1.16 to 8.91 µg
(2.75 ± 1.64 µg). PET data were reconstructed using
filtered back projection, including corrections for
attenuation and scatter (based on a low-dose CT
acquisition). The dynamic data were binned into 26
frames (durations: 8 × 15 seconds, 3 × 1 minutes,
5 × 2 minutes, 5 × 5 minutes, 5 × 10 minutes).
Radioligand synthesis
Radiosynthesis and quality control were performed
on-site, as previously described, obtaining radiochemical purities of >95%.11
[11C]PBR28 PET image and kinetic analysis
T2 FLAIR images were rigidly registered to T1 using
FLIRT (FMRIB Software Library v5.0). WMLs were
manually segmented on the registered T2 image using
Jim software (Xinapse Systems v7). The WML mask
was used for lesion-filling the T1 image before segmentation into white matter, grey matter and cerebrospinal fluid using the FSL (FMRIB Software Library
v5.0) tools FAST and FIRST.19 Normalised brain volumes were calculated using SIENAX (FMRIB
Software Library v5.0).19 A mask of NAWM was created by subtracting the WML mask dilated by 6 mm
around its edges in 3D and the resulting mask further
eroded by 3 mm. The masks of WML, NAWM and
grey matter were multiplied by the mask of the spectroscopy voxel to create the respective masks within
the spectroscopy voxel.
The T1 image and dynamic PET images were used as
inputs for the MIAKAT (Molecular Imaging and
Kinetic Analysis Toolbox) software package (www.
miakat.org) for kinetic analysis of PET data. For this,
PET images were motion corrected using a frame-byframe realignment algorithm, in which all frames
were individually realigned to a reference frame and
rigid registered to the MNI (Montreal Neurological
Institute) space using SPM5 (Wellcome Trust Centre
for Neuroimaging, http://www.fil.ion.ucl.ac.uk/spm).
These transformed four-dimensional (4D) PET
images were integrated over time to obtain 3D PET

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Table of Contents for the Digital Edition of Multiple Sclerosis Journal - October 2017