Patients and controls
A retrospective analysis of the first imaging from patients attending the Sheffield Ataxia Centre was performed. The diagnosis of MSA-C was based on the clinical history in accordance to the published guidelines [1] and neuroimaging findings. SAOA patients were diagnosed by exclusion of identifiable causes. A full history and examination was conducted. Extensive investigations to determine the aetiology of ataxia were performed. Such tests, depending on clinical indications, included blood cell counts, biochemistry, thyroid function, serology for coeliac disease, vitamin A, B12, E; metabolic screen for acyl-carnitines, urinary organic acids, very long chain fatty acids, phytanic acids, lysosymal enzymes, lipoproteins, serum electrophoresis, copper, caeruloplasmin, ferritin and iron, common mitochondrial genetics (MELAS, MERRF, NARP) and in selected cases when clinically indicated, mutations in the mitochondrial DNA polymerase gamma (POLG) gene as well as muscle biopsies. In addition, testing to exclude other ataxia syndromes was performed, including spinocerebellar ataxias (SCA1, 2, 3, 6, 7, 12 and 17) and Friedreich’s ataxia (FA). Additional genetic tests including, for example, DRPLA, fragile X, ataxia occulomotor apraxia, episodic ataxias, SPG7 screening were carried out if clinically indicated. The introduction of next generation sequencing meant that all the ataxia patients in this study underwent further genetic testing on an ataxia gene panel (42 genes) and none were positive.
Patients were excluded if they had identifiable causes of ataxia. The SAOA patients were matched to MSA-C patients for age at first scan. The clinical records were assessed to obtain the clinical symptoms of the patients, the duration of ataxia and severity of the ataxia. The severity of ataxia was divided into three categories by an experienced consultant neurologist as published previously [10, 11]: mild (instability without staggering steps or falls), moderate (instability with staggering steps or falls and/or needs walking aid), and severe (unable to walk despite support from accompanying person).
The MSA-C patients were matched to healthy controls for age at their scan and sex. The healthy controls underwent a thorough screening health questionnaire to ensure they had no form of illness that could result in cerebellar dysfunction.
The project had IRB approval to use patient’s clinical data without written consent. Fully informed written consent was obtained for all healthy control subjects.
Imaging protocol
Scanning was performed using a 3 T system (Philips ACHIEVA 3.0 T Best, Netherlands) with an 8-channel receive only array head coil. The imaging data analysed from patients was from their initial MR scan with us that included our routine imaging protocol for patients with ataxia. The imaging protocol was as follows:
1H-MR spectroscopy
A point-resolved spectroscopy (PRESS) sequence (TR = 2000 ms, TE = 144 ms; 128 measurements; 1024 spectral points; spectral bandwidth 2000 Hz) acquired data at two voxel positions, the superior cerebellar vermis and the deep cerebellar white matter of the right hemisphere, avoiding the dentate nucleus that represents a separate spectroscopy target in our experience. The voxel size was 2.0 × 1.0 × 2.0 cm3. Careful placement of the voxel ensured cerebrospinal fluid contamination of the voxel was minimised. Chemical shift selective imaging pulse technique (CHESS) was used for water suppression. Post processing of the spectra involved zero filling, Gaussian filtering, exponential multiplication, Fourier transform and manual phase correction with baseline subtraction. The quality of the spectra was assessed by two neuroradiologists to ensure adequacy. Figure 1 shows the placement of the voxel in both the cerebellar vermis and hemisphere.
Structural MR imaging
High-resolution three dimensional T1-weighted MR imaging scans were acquired using a magnetisation prepared rapid gradient echo sequence with TR = 11 ms, TE = 4.8 ms, flip angle = 8o, field of view = 256 × 205 × 150 mm, voxel dimension = 0.8 mm (isotropic). Axial T2-weighted images were acquired using a turbo spin echo sequence (TR = 3000 ms, TE = 80 ms; echo train length = 14; 1 NSA, voxel dimension = 0.45 × 0.44 × 4 mm).
Volumetric analysis
FMRIB’s Software Library (FSL) [12,13,14,15,16] was used to perform the structural image volume analysis. The software automatically quantified the volume of the cerebellum and the brainstem and fourth ventricle. Structural Image Evaluation using Normalization of Atrophy cross-sectional method (SIENAX) [17] was used to calculate the brain tissue volume, correcting for subject head size.
The volume of the brainstem and fourth ventricle is calculated as one entity using FSL [18]. The avoid effects of change in the fourth ventricle it was manually segmented on each MR image it was shown on to quantify the volume blinded to whether the images were from a control participant or patient using an extended workstation Philips Medical Systems. The brainstem boundaries were the foramen magnum inferiorly and superiorly by a line drawn across the superior aspect of the midbrain at the level of its interface as the floor of the third ventricle.
Imaging outcomes measures
The following outcomes were measured: (1) brainstem volume expressed as a percentage of total intracranial volume; (2) cerebellar volume expressed as a percentage of total intracranial volume; (3) NAA/Cr and Cho/Cr ratios in the cerebellar hemisphere and vermis. Additionally the presence of the hot cross bun sign was noted.
Statistics analysis
Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) version 20 and MedCalc version 12.3. One way ANOVA was used to compare MSA-C patients with controls and SAOA patients. Post hoc testing was performed by Tukey’s HSD if a significant difference (p < 0.05) was elicited by ANOVA across the study data.