{'fraction_non_alphanumeric': 0.03698845750262329, 'fraction_numerical': 0.022101259181532003, 'mean_word_length': 4.853550863723608, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cross-sectional and longitudinal assessment of the upper cervical spinal cord in motor neuron disease

Background Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease characterized by both upper and lower motor neuron degeneration. While neuroimaging studies of the brain can detect upper motor neuron degeneration, these brain MRI scans also include the upper part of the cervical spinal cord, which offers the possibility to expand the focus also towards lower motor neuron degeneration. Here, we set out to investigate cross-sectional and longitudinal disease effects in the upper cervical spinal cord in patients with ALS, progressive muscular atrophy (PMA: primarily lower motor neuron involvement) and primary lateral sclerosis (PLS: primarily upper motor neuron involvement), and their relation to disease severity and grey and white matter brain measurements. Methods We enrolled 108 ALS patients without C9orf72 repeat expansion (ALS C9–), 26 ALS patients with C9orf72 repeat expansion (ALS C9+), 28 PLS patients, 56 PMA patients and 114 controls. During up to five visits, longitudinal T1-weighted brain MRI data were acquired and used to segment the upper cervical spinal cord (UCSC, up to C3) and individual cervical segments (C1 to C4) to calculate cross-sectional areas (CSA). Using linear (mixed-effects) models, the CSA differences were assessed between groups and correlated with disease severity. Furthermore, a relationship between CSA and brain measurements was examined in terms of cortical thickness of the precentral gyrus and white matter integrity of the corticospinal tract. Results Compared to controls, CSAs at baseline showed significantly thinner UCSC in all groups in the MND spectrum. Over time, ALS C9– patients demonstrated significant thinning of the UCSC and, more specifically, of segment C3 compared to controls. Progressive thinning over time was also observed in C1 of PMA patients, while ALS C9+ and PLS patients did not show any longitudinal changes. Longitudinal spinal cord measurements showed a significant relationship with disease severity and we found a significant correlation between spinal cord and motor cortex thickness or corticospinal tract integrity for PLS and PMA, but not for ALS patients. Discussion Our findings demonstrate atrophy of the upper cervical spinal cord in the motor neuron disease spectrum, which was progressive over time for all but PLS patients. Cervical spinal cord imaging in ALS seems to capture different disease effects than brain neuroimaging. Atrophy of the cervical spinal cord is therefore a promising additional biomarker for both diagnosis and disease progression and could help in the monitoring of treatment effects in future clinical trials.


Introduction
Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease, resulting in upper and lower motor neuron degeneration. It is a heterogeneous disease in terms of disease onset and disease progression (Chiò et al., 2011;Ravits and La Spada, 2009) with a median survival time of 3 years after onset of symptoms (Westeneng et al., 2018). Most in vivo neuroimaging studies in ALS have focused on the brain, showing cortical thinning in several regions including the motor cortex (Agosta et al., 2012;Bede and Hardiman, 2018;Walhout et al., 2015; T Westeneng et al., 2015) and loss of white matter integrity in the structural brain networks of patients with ALS compared to controls (Buchanan et al., 2015;Keil et al., 2012;Schmidt et al., 2014;Verstraete et al., 2011Verstraete et al., , 2014. Furthermore, a different pattern of brain involvement is demonstrated in a subgroup of patients with a C9orf72 mutation (Floeter et al., 2016;Westeneng et al., 2016).
Neuroimaging of the brain, however, targets to find upper motor neuron degeneration. Expanding the focus to spinal cord imaging provides an opportunity to investigate not only upper but also lower motor degeneration (El Mendili et al., 2014. In recent years, spinal cord imaging in ALS has gained more interest (Agosta et al., 2009;Branco et al., 2014;Cohen-Adad et al., 2013;de Albuquerque et al., 2017;El Mendili et al., 2014;Olney et al., 2018;Querin et al., 2017Querin et al., , 2018Rasoanandrianina et al., 2017;Sperfeld et al., 2005;Valsasina et al., 2007;Wang et al., 2014). While significant atrophy of spinal cord was found in patients with ALS compared to controls, it remains unclear whether thinning occurs throughout the motor neuron disease (MND) spectrum or only in ALS, whether thinning differs according to site-ofonset type, and what is the relationship between thinning and grey and white matter degeneration in the brain.
Brain MRI scans include the upper part of the cervical spinal cord allowing us to investigate the effects of motor neuron loss in this area and also enables to link disease effects on cervical cord MRI to those of the brain. Together with ALS, patients with primary lateral sclerosis (PLS), a primarily upper motor neuron disorder, and progressive muscular atrophy (PMA), a primarily lower motor neuron disorder, make up the MND spectrum and studying these patient groups could therefore help in the identification of the type of motor neuron degeneration driving cervical spinal cord thinning. Moreover, spinal cord imaging might help in characterizing disease effects outside the brain, finding relationships with phenotype, genotype and disease severity, and as a diagnostic and prognostic biomarker. Finally, longitudinal neuroimaging studies have the potential to monitor disease progression and could be used as biomarker in clinical trials.
In the present study, we investigated cross-sectional and longitudinal disease effects in a large cohort of patients with ALS, PLS or PMA using neuroimaging of the upper cervical spinal cord derived from brain MRI.

Participants
Patients with a MND were recruited from a population-based cohort shortly after diagnosis (Huisman et al., 2011), between 2009 and 2018. ALS patients were diagnosed according to the El Escorial criteria (Brooks et al., 2009). PLS patients were diagnosed according to the Gordon criteria, where the patient fulfilled the criteria at baseline or during follow-up (de Vries et al., 2018;Gordon et al., 2006). For the latter, earlier scans were included in the analyses. Similarly, patients with PMA satisfied previously described criteria from literature (de Vries et al., 2018;Visser et al., 2007). Clinical characteristics such as site of and age at disease onset, and the revised ALS functional rating scale (ALSFRS-R) score as measure for disability (Cedarbaum et al., 1999) were recorded. The same protocol was used to acquire longitudinal data. Genomic DNA samples of patients were screened for the C9orf72 repeat expansion, using a repeat primed PCR as previously described (Van Rheenen et al., 2012). Patients have not been screened routinely for other ALS-related mutations as these are relatively rare. Patients had no history of brain injury, psychiatric illness, epilepsy, or neurodegenerative diseases other than ALS or frontotemporal dementia. Control subjects were also selected from a Dutch population- Fig. 1. Cervical spinal cord segmentation. 1) A control subject was appointed as template for spinal cord segmentation. 2) The initial segmenting starting point derived on the template was brought to individual space and used to segment the spinal cord of each subject. 3) For each subject, borders of the cervical spinal cord segments were identified as the center of intervertebral disks. 4) The cross-sectional area (CSA) of each segmentation slice was computed and averaged over the first three cervical segments (UCSC) or for individual segments (C1-C4). based cohort (Huisman et al., 2011). These controls are, therefore, not necessarily healthy but are controls in the sense that they have no MND or a history of brain injury, psychiatric illness or epilepsy. Participants (patients and controls) with cervical myelopathy, cervical radiculopathy, spinal trauma or spinal surgery of the studied spinal segments, and subjects with demyelinating conditions (multiple sclerosis / neuromyelitis optica) were also excluded. The Ethical Committee for human research of the University Medical Center Utrecht approved the study protocols and informed written consent, according to the Declaration of Helsinki, was obtained from each subject.

Image acquisition
From all subjects, T1 scans were acquired using a 3 Tesla Philips Achieva Medical Scanner with a SENSE receiver head-coil, initially for the purpose of brain imaging. Each high-resolution T1-weighted image was acquired by a 3D fast field echo using parallel imaging (acquisition time = 11 min, TR/TE = 10/4.6 ms, flip-angle 8°, slice orientation: sagittal, voxel size = 0.80 × 0.75 × 0.75 mm, field of view (FOV) = 176 × 240 × 240 mm, gradient nonlinearity correction was applied), covering the whole brain and the first segments of the cervical spinal cord (C1-C2). The proportion of other cervical spinal cord segments (C3-C5) present on the images depended on the subject's head size and position in the field of view, while the subject's head and upper cervical spinal cord (UCSC) was consistently positioned in the middle of the head coil.
Furthermore, diffusion-weighted images (DWI) of the brain were acquired to assess white matter. For this, two sets of 30 diffusionweighted scans and five unweighted B0 scans each were acquired as follows: DWI-MR using parallel imaging SENSE, p-reduction = 3, gradient set of 30 different weighted directions, b = 1000 s/mm 2 , TR/ TE = 7035/68 ms, 2 × 2 × 2 mm voxel size, 75 slices, second set with reversed k-space read-out, acquisition time = 4.5 min. A) The CSA of UCSC for each patient group (PLS, ALS C9-, PMA and ALS C9+) was compared to their age-and gendermatched controls (CON). Solid lines denote the mean CSA and dashed lines reflect the mean ± standard deviation boundaries. B) Comparison between patient and control groups for cervical segments C1-C4.

Spinal cord data processing
Using the T1-weighted images, we identified a control subject with minimal anterior-posterior and left-right curvature of the spine and the most cervical segments (C1-C5). This subject was set to be the experimental standard template, following the framework of Kong et al. (2014).
The spinal cord of the template was segmented, using segmentation functions of the Spinal Cord Toolbox , by manually setting the initial segmenting starting point for the template (Fig. 1).
The T1-weighted image of each subject was registered to the template by applying FSL's non-linear image registration algorithm (FNIRT), which used the output of the linear registration algorithm  (FLIRT) as affine starting guess. From this registration, an inverse transform matrix was obtained, which, in turn, was applied to bring the segmentation starting point of the template to individual subject space. Next, the spinal cord of all subjects was segmented using the Spinal Cord Toolbox based on the derived initial starting point as input in order to optimize the performance of these segmentation functions.
Each segmentation was visually checked and, if necessary, improved manually (blinded for disease status). Finally, the centers of intervertebral disks served as the borders dividing the cervical spinal cord segments and were identified for all subjects (Fig. 1).

Brain data processing
Cortical and subcortical brain regions were analyzed using T1 images and FreeSurfer (V5.3.0, http://surfer.nmr.mgh.harvard.edu/). In total, 83 distinct brain regions (68 cortical regions, 14 subcortical regions and brainstem) were parcellated and segmented (Fischl et al., 2002(Fischl et al., , 2004 according to the Desikan-Killiany atlas (Desikan et al., 2006). From the DWI dataset, white matter integrity was assessed. Preprocessing included correction for susceptibility and eddy-current distortions, as previously described (Verstraete et al., 2011). Next, the images were processed with the TRACULA tool (TRActs Constrained by Underlying Anatomy) for automated reconstruction of major white matter tracts, like the corticospinal tract (CST), using global probabilistic tractography (Yendiki et al., 2011). The output included fractional anisotropy (FA), radial diffusivity (RD) and mean diffusivity (MD) values for the major white matter tracts.

Cross-sectional area
We calculated the mean cross-sectional area (CSA) of the upper cervical spinal cord (UCSC), defined as the mean of all slices in the first three spinal cord segments (i.e. C1-C3, Fig. 1) with CSA of each segmentation slice computed using the sct_process_segmentation function of the Spinal Cord Toolbox . This function counts pixels in each slice, multiplies them by the voxel area and then geometrically adjusts this using the centerline orientation of the spinal cord to obtain the CSA. Next, we investigated disease effects in individual cervical segments, defined by the identified borders, and computed the mean CSA over all slices in each segment (Fig. 1). For this analysis, we also included segment C4 as the CSA in this segment could be calculated for at least 25% of subjects in each patient group. In contrast, C5 was usually (90%) outside the field of view, so we constrained the segmentwise comparison to C1-C4.

Statistical analysis
Statistical analyses were executed using MATLAB Release 2017b, including the Statistics Toolbox (The MathWorks, Inc., Natick, Massachusetts, United States; http://www.mathworks.com). We matched each patient to an age-and gender-matched control subject. For this, we first divided the patient and control groups according to gender. For each gender-group, we then calculated the Euclidean distance based on age between each patient and the controls. The control subject with the smallest distance was chosen as the match for this patient and was not available anymore for the matching algorithm of this patient subgroup.
First, baseline cross-sectional analyses were performed. Patient groups were compared to their age-and gender-matched controls based on their upper spinal cord and segment-wise measurements. No direct comparisons between patient subgroups were performed due to the dependence of disease duration, different sample sizes of the groups, and because we focused primarily on 'abnormality' (thus compared to controls). We therefore restricted the analyses to comparisons with age-and gender-matched controls.
Significance between groups was assessed by means of linear models (age and gender were used as covariates) and permutation tests in which group labels were randomly assigned (10,000 permutations). P-values < 0.05, after FDR-correction for multiple testing, were considered statistically significant. We used the algorithm of Benjamini and Hochberg for this FDR-correction (Benjamini and Hochberg, 1995).
Second, we conducted longitudinal analyses using linear mixed-effects models to assess CSA change over time. Age and gender were included as covariates and we accounted for random between-subject variation of slopes and intercepts. To examine disease effects and to compare whether one group shows faster deterioration than others, we included an interaction term (follow-up time × group), where follow-up time was defined as the time since baseline scan at follow-up scan, and group referred to patient or control. The reported values reflect the greater amount of CSA that a patient group has lost per month compared to the control group.
Third, we investigated CSA in relationship to phenotype, where we stratified the C9orf72-negative ALS patient group (ALS C9-) for their site of disease onset (bulbar or spinal). Furthermore, the CSAs of patients were correlated with their total ALSFRS-R scores and their total ALSFRS-R minus the bulbar sub-score (spinal ALSFRS-R sub-score (Rasoanandrianina et al., 2017)), obtained at the time of scan, to link the spinal cord measurements to disease severity. For the longitudinal MRI and ALSFRS-R measurements, a linear model with random slopes and intercepts was computed. Finally, we investigated a possible link between brain measurements (i.e. cortical thickness of precentral gyrus and FA of the CST) and CSA.

Participants
In total, 108 ALS C9-patients and 26 ALS patients with a C9orf72 mutation (ALS C9+), 28 PLS and 56 PMA patients participated in the study (Table 1 and S1). In addition, 114 unrelated population-based controls were included in the study and matched based on age and gender to each of the patient groups (Table S2). Follow-up data (up to five visits, Table S3) were available for 64 ALS C9-, 18 ALS C9+, 18 PLS and 41 PMA patients. Of the total control group of 114 subjects, 54 controls had two visits (i.e. 51 controls matched to the ALS C9-group, 11 controls for the ALS C9+ group, 12 controls for the PLS and 25 controls for the PMA patients). CSA of segments C1 and C2 were available for all participants (Table S4). The CSA of C3 and C4 could be calculated for at least 75% and 25% of the participants in each subject group, respectively.
ALS C9-and PLS patients showed differences in CSA compared to their controls for all individual segments of the cervical spinal cord (C1-C4, Fig. 2B, Table 2). The CSA of the PMA group were significantly lower than the controls at level C1-C3. Finally, the ALS C9+ patients displayed thinner segments compared to control at level C2 and C3.
Patients with and without follow-up data displayed similar CSA results at baseline, with no significant difference between the two groups (Fig. S1). Averaging only segments C2 and C3 instead of UCSC (i.e. C1-C3) resulted in similar findings as for UCSC (Table S5).
Compared to controls, significant thinning of C3 was observed over time in ALS C9-patients, but no longitudinal changes in segments C1, C2 and C4 (Table 3). Progressive thinning over time was also observed in C1 of PMA patients, whereas the spinal cord thickness of the PLS and ALS C9+ group did not change over time in any of the examined segments.

Relationship with site of onset
In ALS C9-patients, both the bulbar-(n = 30) and spinal-onset (n = 78) patients showed a significantly reduced mean UCSC CSA compared to controls (bulbar onset: mean ΔCSA = −4.12 mm 2 , 95%-CI [−7.19;−1.04], p = 0.022; spinal onset: mean ΔCSA = −4.33 mm 2 , 95%-CI[−6.83;−1.83], p = 0.003); no significant difference was found between the two groups ( Fig. 4A and Table 4). Both onset types displayed a significantly smaller mean CSA in segment C3 compared to controls, where spinal-onset patients additionally revealed significantly smaller CSA at the level of C2 (Fig. 4B and Table 4). Again, there were no significant differences between the separate segments when comparing the bulbar and spinal onset groups directly.

Relationship with ALSFRS-R
The total ALSFRS-R score correlated significantly with the upper spinal cord measurement of PLS patients (r = 0.557, p = 0.009, Fig. 5A) and the first three segments separately (C1: r = 0.547, p = 0.003; C2: r = 0.541, p = 0.003; C3: r = 0.463, p = 0.034; C4: r = 0.349, p = 0.396). Of note, the correlation for C4 was based on only eight subjects, so insufficient power could explain the lack of a significant correlation with the total ALSFRS-R score in this segment. In the other groups, we observed no significant correlations between the upper spinal cord measurement and the total ALSFRS-R score, nor for the individual segments (Table S7).
Similar results were found for the spinal ALSFRS-R sub-score in the various groups for both the cross-sectional and longitudinal analyses (Tables S7 and S8).

Relationship with cerebral motor system
In contrast to the UCSC result, only the ALS C9-and ALS C9+ patients displayed a significantly thinner motor cortex of the precentral gyrus (averaged over both hemispheres, Fig. 6A) compared to their matched controls at group-level. However, correlating CSA with cortical thickness of the precentral gyrus did not reveal a significant relationship (Table 6).
For the cerebral part of corticospinal tract, the ALS C9-patients showed significantly lower FA than their matched controls, whereas the other patient groups did not (Fig. 6B). The MD of the CST was significantly increased for all but PMA patients and we only found significantly increased RD of the CST for ALS C9- (Table S9). The PLS and PMA groups displayed a significant correlation between FA of the CST and CSA, both for UCSC and separate segments, although the direction of the correlation was opposite between these two groups (Table 6). We found similar results when using RD or MD as white matter integrity measure for the CST. Only for ALS C9+, other major white matter tracts reached significance (Table S9).

Discussion
In the present study, we investigated the value of upper cervical spinal cord measurements as biomarker of diagnosis and disease progression in patients with ALS, and its upper motor neuron variant, PLS, and lower motor neuron variant, PMA. We showed a thinner upper cervical spinal cord compared to controls in all disease groups (ALS, PLS and PMA), which might suggest that cervical spinal cord thinning is related to both upper and lower motor neuron loss. Interestingly, patients with a C9orf72 repeat expansion, who have an MRI phenotype   (Bede et al., 2013;Westeneng et al., 2016), did not show such widespread significantly thinner cervical spinal cord at baseline. Over time, significant thinning occurred in the ALS patient group without C9orf72 repeat expansion, while the PMA patients only show thinning in one segment (C1), and ALS C9+ as well as PLS patients displayed no significant longitudinal thinning at all. Longitudinal cervical spinal cord imaging might, therefore, be a valuable biomarker of disease progression in clinical trials. Several previous studies showed no differences in spinal cord area or diameter between ALS patients and controls at the level of C2-C4 (Sperfeld et al., 2005;Wang et al., 2014). Other research revealed a thinner spinal cord in these regions, applying 1.5 T or 3 T MRI in up to 64 patients (Agosta et al., 2009;Branco et al., 2014;de Albuquerque et al., 2017;Olney et al., 2018;Rasoanandrianina et al., 2017;Valsasina et al., 2007). Differences in results might be explained by the use of 1.5 T or 3 T MRI scanner, different study population sizes or the measurements of only one slice in a segment, a mid-vertebra slice, or an average over slices in entire segments. Compared to studies that also found thinner spinal cord cross-sectionally, we demonstrated smaller area differences (Branco et al., 2014;de Albuquerque et al., 2017;Olney et al., 2018;Valsasina et al., 2007). Disease duration of the patients at baseline might also influence the CSA differences: the median disease duration of the ALS patients in our population is shorter than in study populations of previous research. Therefore, our cross-sectional findings might reflect an earlier phase of upper cervical spinal cord degeneration since our longitudinal analyses also showed growing differences over time. We demonstrated in a large well-characterized, C9orf72-genotyped cohort of patients, thinner spinal cord in MND patients, by both examining the mean CSA in UCSC and individual segments. In this way, we obtained a robust measure of spinal cord thickness, while still considering the possibly variable behavior of the disease at different levels of the cervical spinal cord. We anticipate applying these analyses to MRI of the entire spinal cord also including thoracic and lumbosacral segments in future studies.
Over time, we demonstrated thinning of the UCSC in ALS C9-patients, which is in line with previous studies (Agosta et al., 2009;de Albuquerque et al., 2017;El Mendili et al., 2014). We found no significant longitudinal change in the UCSC for the other patient groups. This might be explained by a relatively more substantial decrease of CSA in controls in these groups (reductions of 2.38 mm 2 /year and 1.54 mm 2 /year for controls matched to ALS C9+ and PLS, compared to reductions of 1.08 mm 2 /year and 0.98 mm 2 /year for controls matched to ALS C9-and PMA). This substantial decrease in controls could be due to smaller group sizes. The similar reduction of the CSA that seems to occur for ALS C9+ and ALS C9-patients supports this explanation. Comparing these decreases of CSA in controls to literature (Casserly et al., 2018;de Albuquerque et al., 2017), they are higher than expected (Fig. S3) and could be caused by our study design: although subjects with spinal cord diseases were excluded, controls were not necessarily healthy as they were recruited from a population-based cohort to reduce potential selection bias. Overall, the small sample sizes and the noteworthy decrease of CSA in controls might potentially create a bias in the longitudinal results. In specific segments, thinning over time was seen for all patient groups except ALS C9+ and PLS. The absence of significant thinning over time for the PLS group might be explained by the longer disease duration at baseline in these patients. Thinning of upper spinal cord in ALS patients in our study was not driven by site of onset. The smaller group of patients with bulbar onset could explain the absence of significant thinning for this group over time as the slope of CSA reduction per month is similar to the slope in spinal-onset patients. A difference between the site-of-onset groups might be revealed more pronounced when examining lower segments of the (cervical) spinal cord. For example, patients with upper-limb onset seem to have a distinct metabolic activation in cervical segments compared to lower-limbonset patients (Marini et al., 2018).
We could not reveal significant correlations between CSA and the ALSFRS-R total score, with or without bulbar sub-scores, for the ALS patients at baseline, and thus could not demonstrate a significant relationship between spinal cord thickness and functional disability at this time point. Previous results concerning this aspect have been conflicting: some studies did (Branco et al., 2014;El Mendili et al., 2014) and others did not find a significant correlation (Agosta et al., 2009;Valsasina et al., 2007). On the other hand, we did find a significant correlation between CSA and the ALSFRS-R score for patients with PLS, both for UCSC and individual segments, possibly reflecting a link between functional disability and pure upper motor neuron loss. Importantly, we did find a relationship between the longitudinal measurements of CSA and total ALSFRS-R score, indicating that disease progression is reflected in the cervical spinal cord.
Although we obtained brain and spinal cord measurements from the same image, we could not display a direct relationship between these measurements for the ALS patients. The lack of this direct relationship might be due to the progressive or multifocal pathogenic nature of ALS, possibly leading to different disease effects (of both upper and lower motor neurons) in brain and spinal cord which level out when correlating the two sites. Possible support for this hypothesis is the fact that this relationship was found between spinal cord and the white matter integrity of the CST for PLS and PMA patients. In PLS patients we demonstrated that a thinner spinal cord also correlated with white matter integrity being more affected. The positive correlation for the PLS patients was in line with our expectations and might possibly support UMN involvement in the upper cervical spinal cord. In contrast, the correlation for PMA patients was inverted: more affected white matter integrity implied a thicker spinal cord. This negative correlation for PMA is harder to explain. One might hypothesize the occurrence of a protection mechanism in the upper cervical spinal cord for the PMA patients (preventing them of becoming ALS patients), which needs to be investigated in future research.
The upper cervical spinal cord is a challenging area to image with MRI. Since we have used an MRI protocol initially for the purpose of brain imaging, we analyzed a region at the edge of the FOV. CSA measurements could, therefore, be influenced by intensity drop or gradient nonlinearity (Stroman et al., 2014). However, the MRI images used in our study had a homogeneous signal intensity across the FOV (see Fig. S2 for example images) and were corrected for gradient nonlinearity. Although there still might be measurement errors present in such a small structure as the UCSC, this would affect both patients and controls equally and, therefore, the observed differences in CSA may still be regarded as disease effects.
In this study, we focused on the upper part of the cervical spinal cord that was included on brain MRI. In support of our method, previous research has successfully shown differences in the cervical spinal cord using brain MRI (de Albuquerque et al., 2017). We also revealed disease effects in the upper cervical spinal cord, but caution is required when interpreting significant results at the level of C4, as analyses of this segment could be applied only to a relatively small group in our study. Investigation of the entire spinal cord would provide a complete overview of atrophy in this important structure. However, as all central motor nerves innervating the upper and lower limbs pass through the upper cervical spinal cord, a correlation with motor deficits could be revealed on the basis of this upper part of the spinal cord. We could not include specific measures for white and grey matter atrophy as the contrast between grey and white matter in the upper cervical spinal cord on our T1-weighted images was not sufficient enough. Future research should include DTI measures and T2-or T2*-weighted MRI to obtain a comprehensive view of grey and white matter damage in the upper cervical spinal cord (Martin et al., 2017;Stroman et al., 2014). Furthermore, test-retesting of the method used in this study and other reliability tests is of importance before upper cervical spinal cord MRI can be used as a biomarker in clinical trials.
In this study, we showed differences between patients and controls in cervical spinal cord MRI, suggesting its potential as diagnostic and longitudinal biomarker in the spectrum of MND.

Disclosure
This work was supported by the ALS Foundation Netherlands. MPvdH was further supported by the Netherlands Organization for Scientific