1. GRAY MATTER ATROPHY IN PEDIATRIC MS PATIENTS
E. De Meo1,2, M.A. Rocca1,2, E. Pagani1, A. Ghezzi3, B. Colombo2, M.E. Rodegher2, L. Moiola2, P. Veggiotti4, R. Capra5, M.P. Amato6, A. Fiorino2, L. Pippolo3, M.C. Pera4, G. Comi2,
A. Falini7, M. Filippi1,2
1Neuroimaging Research Unit, Institute of Experimental Neurology, Division of Neuroscience, 2Dept. of Neurology, and 7Dept. of Neuroradiology, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy; 3MS Center, Ospedale di Gallarate, Gallarate, 4Dept. of Child Neurology and Psychiatry, C. Mondino National Neurological Institute, Pavia, 5MS Center, Spedali Civili of Brescia, Brescia, 6Dept. of Neurology, University of Florence, Florence, Italy.
Partially supported by grants from Italian Ministry of Health (GR ) and Fondazione Italiana Sclerosi Multipla (FISM2011/R/19 and FISM 2012/R/8).

2. Background
In multiple sclerosis (MS), the gray matter (GM) is involved from the earliest phases of the pathological processes;
Previous cross-sectional MRI studies have shown that also pediatric MS patients have reduced thalamic (Mesaros et al., Neurology 2008) and brain volumes (Aubert-Broche et al., Neuroimage 2011) compared to age- and sex-matched healthy controls (HC);
To date, only one longitudinal study has been conducted in pediatric MS patients concluding that onset of MS during childhood limits age-expected primary brain growth and leads to subsequent brain atrophy (Aubert-Broche et al., Neurology 2014).

3. Background
Weier et al., MSJ 2016
MonoADS, ADEM, and MS in childhood lead to impaired age-expected growth of the cerebellum;
Cerebellar volume differences between HC and patient groups were most evident in the posterior lobes.
Aubert-Broche et al., Neurology 2014

4. Objectives Experiment (1) Experiment (2)
To define the regional patterns of GM atrophy progression in pediatric MS patients;
To explore the correlations with clinical and conventional MRI measures.
Experiment (2)
To investigate pathobiological basis of the different clinical course between pediatric and adult MS patients;
To compare brain atrophy development between pediatric and adult MS patients;
To explore whether and how the atrophy patterns detected correlate with clinical and conventional MRI measures.

5. Study subjects Subjects (1) *Pearson’s chi square test HSR HC NIH HC
Pediatric MS patients
p values
Number of subjects 26
119 31 -
Female/male
13/13
64/55
19/12
0.45*
Mean age (SD) [years]
15.2
( )
14.9
( )
15.5
( )
0.72
Median disease duration (range) [years]
1.29
( )
Median EDSS [range]
1.0
( )
Mean T2 LV (SD) [ml]
4.9 (6.5)
Mean T1 LV (SD) [ml]
2.7 (3.5)
*Pearson’s chi square test

6. Study subjects Subjects (1) Subjects (2)
HSR HC
NIH HC
Pediatric MS patients
p values
Number of subjects 26
119 31 -
Female/male
13/13
64/55
19/12
0.45*
Mean age (SD) [years]
15.2
( )
14.9
( )
15.5
( )
0.72
Median disease duration (range) [years]
1.29
( )
Median EDSS [range]
1.0
( )
Mean T2 LV (SD) [ml]
4.9 (6.5)
Mean T1 LV (SD) [ml]
2.7 (3.5)
Pediatric HC
Adult HC
Pediatric MS patients
Adult MS patients
p values
Number of subjects 26 30 31 -
Female/male
13/13
17/13
19/12
18/12
n.s.§*
Mean age (SD) [years]
15.2
( )
35.1
( )
15.5
( )
34.77
( )
n.s.
Median disease duration (range) [years]
1.29
( )
4.32
( )
0.001
Median EDSS [range]
1.0
( )
1.5
( )
Mean T2 LV (SD) [ml]
4.9 (6.5)
4.6 (3.9)
Mean T1 LV (SD) [ml]
2.7 (3.5)
3.3 (3.4)
*Pearson’s chi square test
§Results of comparison between age matched study groups.
*Pearson’s chi square test
Longitudinal scans at a mean follow-up of 3.5 years were available from pediatric MS patients, NIH HC and adult MS patients;
Both pediatric and adult MS patients underwent to clinical evaluation at baseline and at follow-up.

7. MRI acquisition
MRI Acquisition (3.0 T scanner) pediatric and adult MS patients and HC from San Raffaele Hospital:
Dual-echo TSE;
3D T1-weighted fast field-echo scan.
MRI Acquisition (1.5 T scanner) Pediatric NIH HC:
Conventional MRI analysis:
Measurements of T2 hyperintense and T1 hypointense lesion volumes (LV) (Jim 6 software);
Quantification of normalized brain (NBV), white matter (WMV) and GMV volumes (SIENAx).

8. Methods MRI analysis (SPM12 software):
Voxel Based Morphometry (to assess regional GM differences at baseline):
Segmentation into GM, WM and CSF;
GM and WM segmented images of all subjects were used to produce GM and WM templates and to drive the deformation to the templates;
Affine registration between the customized GM template and the SPM GM template in the Montreal Neurological Institute (MNI) space.
Tensor Based Morphometry (to assess regional pattern of GM atrophy progression):
Pairwise longitudinal registration to align the first and second scan of each subject;
Volume change quantification;
Production from the GM and WM segmented images of GM and WM templates;
Normalization to MNI space.

9. Statistical analysis
Two sample t test to assess GM differences at baseline:
Experiment (1): HSR HC vs NIH HC, MS vs HSR HC and MS vs NIH HC ;
Experiment (2): pediatric HC vs adult HC, pediatric MS vs pediatric HC, pediatric MS vs adult MS, adult MS vs adult HC;
One sample t test to assess longitudinal GM changes within group:
Experiment (1): NIH HC and pediatric MS;
Experiment (2): pediatric MS and adult MS;
Analysis of covariance (ANCOVA) to compare longitudinal GM volume changes between-groups:
Experiment (1): MS vs NIH HC;
Experiment (2): pediatric MS vs adult MS;
Multiple regression models were used to asses correlations between GM volume changes and clinical and conventional MRI variables.

10. Results / Experiment 1 Baseline NIH HC > Pediatric MS
HSR HC > Pediatric MS
(two-sample t test, p<0.05 FWE corrected)
No significant differences of the regional distribution of GM volumes between HSR HC and NIH HC were found;
Compared to NIH HC, pediatric MS patients showed thalamic atrophy, bilaterally; 
Compared to our cohort of HC, pediatric MS patients showed thalamic atrophy, bilaterally;
No areas of increased GM volume were detected in pediatric MS patients, compared to both groups of pediatric HCs.

11. Pediatric MS patients> Adult MS patients
Results / Experiment 2
Baseline
Compared to age-matched HC, adult MS patients showed a distributed pattern of GM atrophy, involving the bilateral thalamus, hippocampus, cingulate cortex and several cortical areas in the frontal, temporal and parietal lobes.
T values P A R L
Pediatric MS patients> Adult MS patients
No areas of greater reduction of GM volume were found in pediatric compared to adult MS patients;
The decreased thalamic volume detected both in pediatric and adult MS patients, when compared to age-matched healthy controls, correlated with T1 and T2 LV.

12. Results / Experiment 1 Longitudinal changes Correlation analysis
No correlations were found between GM atrophy development and disease duration, age at onset and EDSS scores (as assessed at baseline, at FU and its changes over time).
Significant correlation was found between thalamic atrophy and T2 and T1 LV at FU (right thalamus: r=0.76; p<0.001; left thalamus: r=0.69; p<0.001).

13. Results / Experiment 2 Longitudinal changes
A similar pattern of GM atrophy progression was found in adult and pediatric MS patients;
Less atrophy in bilateral temporal pole, left insula, hippocampus, middle frontal gyrus and cerebellum was found in adult MS patients;
No significant correlations were found between atrophy progression in adult MS patients and T1 and T2 LV as estimated both at baseline ad FU.

14. Conclusions
In line with previous cross-sectional studies, at baseline we found reduced thalamic volume in pediatric MS patients compared to matched HCs;
The regional analysis of longitudinal volume changes showed a failure of the age-expected GM trajectories of development in patients with pediatric MS;
In addition to the age-expected growth failure, significant atrophy and continuous progression of atrophy were found at the level of the thalamus, suggesting that the reduced volume of this structure might result from both a failure of age-expected brain growth as well as a subsequent and progressive reduction in established brain volume.

15. Conclusions
The similar pattern of GM atrophy progression detected between adult and pediatric MS patients suggested that the increased resilience of pediatric patients, as demonstrated by the increased myelin repair capabilities does not effective counteract GM involvement;
Our findings confirm that neurodegeneration represent an early aspect of MS pathology, only partially related to inflammation, rather than a late effect of chronic disease, emphasizing the importance of implementing neuroprotective strategies in order to prevent the accrual of disability starting form the earliest phases of disease.