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Subcortical grey matter volumes predict subsequent walking function in early multiple sclerosis
Journal of the Neurological Sciences 366 (2016) 229–233
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Subcortical grey matter volumes predict subsequent walking function in early multiple sclerosis Bardia Nourbakhsh a,⁎, Christina Azevedo c, Amir-Hadi Maghzi d, Rebecca Spain e, Daniel Pelletier c, Emmanuelle Waubant a,b a
Department of Neurology, University of California San Francisco, San Francisco, CA, United States Department of Pediatrics, University of California San Francisco, San Francisco, CA, United States Department of Neurology, University of Southern California, Los Angeles, CA, United States d Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, United States e Department of Neurology, Oregon Health and Science University, Portland, OR, United States b c
a r t i c l e
i n f o
Article history: Received 13 January 2016 Received in revised form 4 April 2016 Accepted 28 April 2016 Available online 06 May 2016 Keywords: Multiple sclerosis Grey matter Ambulation Atrophy
a b s t r a c t Background: Atrophy of subcortical grey matter structures has been reported to be associated with clinical measures of disability in multiple sclerosis (MS) patients. It is not clear if the degree of tissue loss in patients with very early MS is associated with changes in disability measures. Objective: To study the association between subcortical grey matter structure volumes and clinical disability outcomes. Methods: Relapsing MS patients within 12 months of clinical onset were enrolled in a neuroprotection trial of riluzole versus placebo with up to 36 months of follow-up and serial brain MRI and clinical assessments. MRI metrics, including thalamic, putamen, caudate, pallidum and cerebellar cortical volume, were measured by an automated, custom-made FreeSurfer pipeline. Volumes were normalized for head size. Clinical measures included EDSS, MSFC scores and its components. Mixed model regression measured time trends and associations between imaging and clinical outcomes. Results: 42 patients with a mean follow-up of 30.6 months were analyzed in this study. There was a statistically significant decrease in thalamus, caudate and putamen volumes, but not cerebellar cortical and pallidum volumes during the follow-up period. Baseline thalamus, caudate and putamen volumes predicted subsequent changes in the timed 25-ft walk test (p = 0.036) and MSFC (p = 0.024). There was a trend for an association between baseline caudate volume and subsequent change in the timed 25-ft walk test (p = 0.084). No association between baseline imaging and subsequent EDSS changes were seen. Conclusion: Subcortical grey matter volumes at early stages of MS are associated with subsequent changes in disability measures. © 2016 Elsevier B.V. All rights reserved.
1. Introduction MS has been traditionally defined as an inflammatory, demyelinating disease affecting the white matter in the central nervous system (CNS); however, grey matter pathology is increasingly recognized to occur early and throughout the disease course, and is associated of irreversible disability [1]. Grey matter pathology can be evaluated by different methods, including studying grey matter lesions, volumetric changes or assessment ⁎ Corresponding author at: Department of Neurology, University of California San Francisco, 675 Nelson Rising Lane, Room 221F, San Francisco, CA, Box 3206, 94158, United States. E-mail address: [email protected] (B. Nourbakhsh).
http://dx.doi.org/10.1016/j.jns.2016.04.054 0022-510X/© 2016 Elsevier B.V. All rights reserved.
of diffusion, magnetization transfer or spectroscopic changes [2]. Atrophy of the grey matter structures in the brain and spinal cord is a surrogate for neuronal loss. It better correlates with or predicts reaching disability milestones as compared to the markers of white matter injury [3]. Grey matter atrophy has been reported in patients within earliest stages of MS. [4–7] It has also been shown to correlate with physical disability [6,8,9]. However; this association has been shown in crosssectional studies and in patients with long-standing disease. Studying neuronal loss and its relevance to clinical measures of performance and disability will help design future neuroprotection trials in early MS which might be an ideal time for intervention. Our aim was to study the cross-sectional and longitudinal associations of brain deep grey matter structures and disability outcomes as measured by Expanded Disability Status Scale (EDSS), timed 25-ft walk (T25FW), 9-hole peg
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test (9HPT) and paced auditory serial addition test (PASAT) in very early MS. 2. Methods We analyzed data from a 2-year randomized double blind, placebo controlled trial of riluzole vs placebo. Details of the study, inclusion and exclusion criteria and the report of primary and secondary endpoints have been previously reported [10]. Relapsing-remitting and clinically isolated syndrome (CIS) patients within 12 months of symptom onset were offered participation in the trial. The first half of patients who completed the 24-month core study were offered continuation for another 12 months (mean follow-up of 30.6 months). Three months after randomizations, all patients started weekly interferon beta-1a. The study was approved by the institutional review boards of the participating centers (UCSF, OHSU). Informed consent was obtained from all participants before enrollment. Because riluzole, as compared to placebo did not show any significant effect on the primary and secondary outcomes of the study (including no effect on the clinical measures or grey matter structures), we combined both treatment groups for the analyses of the associations.
hypothesis testing. All analyses were conducted in Stata Version 13.1 (Stata Corp, College Station TX).
3. Results 3.1. Patient's characteristics Baseline demographic, clinical and radiographic characteristics of patients who were recruited in the study are shown in Table 1. Forty two of 43 patients enrolled in the clinical trial contributed to the longitudinal assessment of the association between MRI and clinical measures. MRI images of one patient could not be processed by the FreeSurfer. Only 5 patients (2 in the riluzole group and 3 in the placebo group) switched to another disease-modifying medication during the study, as treating physicians determined there was clinical or radiological disease activity on intramuscular weekly IFN-beta 1a. Relapse rate was 0.22 per year in the placebo and 0.15 in the riluzole group (p = 0.27).
3.2. Longitudinal changes in grey matter structures
EDSS and MSFC [including PASAT3′] were used to monitor neurological changes [11,12]. Patients had screening and baseline MSFC to allow for practice effects. The screening MSFC was not used in the analyses.
During the study, there was a statistically significant decrease in the volume of thalamus (1.4% per year, 95%CI: 0.6%–2.3%, p = 0.001), caudate (2.8% per year, 95%CI: 2.1%–3.5%, p b 0.001) and putamen (2.5% per year, 95CI: 1.5%–3.4%, p b 0.001), but not cerebellar cortical and pallidum volumes (Fig. 1 and Table 2).
2.2. MRI acquisition and post-processing
3.3. Longitudinal changes in the clinical measures
Details of the MRI protocol have been described previously [10]. Brain MRIs were performed on 3.0 Tesla MRI scanners equipped with an 8-channel phased array coil (General Electric, Milwaukee, WI) at baseline and months 6, 12, 18, 24 and for half of patients, at month 36. The standardized study protocol included a 3D, T1-weighted, volumetric, 1 mm-isotropic inversion recovery spoiled gradient-echo sequence (3D-IRSPGR, 1 × 1 × 1 mm3, 180 slices), which was used for all brain volume measurements, as well as a 2D multislice dual spin echo sequence (proton density and T2-weighted, 1 mm × 1 mm × 3 mm, no gaps). T1 lesion masks were used to inpaint 3D-IRSPGR images prior to submission to FreeSurfer [13] to avoid voxel misclassification errors. Anatomic segmentation was performed using FreeSurfer's longitudinal processing stream (v5.3; probabilistic atlas for subcortical segmentation) [13]. FreeSurfer's output was reviewed, manually corrected and reran as needed by an experienced MRI postprocessor. Deep grey and cerebellar cortical volumes were extracted directly from FreeSurfer's output and normalized for head size using the estimated total intracranial volume, also taken from FreeSurfer. The resulting values are unitless.
During the study, there was a statistically significant worsening in EDSS (0.18 point/year, 95%CI: 0.07–0.28, p = 0.005). There was no statistically significant longitudinal change in other clinical measures over time.
2.1. Clinical measurements
2.3. Statistical analysis Descriptive statistics for patient characteristics were presented either as percentages (%) or using mean ± standard deviation (SD). Spearman correlation was used to assess the cross-sectional association of clinical and imaging outcomes at baseline. Mixed effects regression models were used to account for the longitudinal nature of the data. To separate the between-participant from the within-participant effects, the baseline values and change from the baseline values of imaging markers were entered in the model as predictors [14]. We performed adequate model checking, including evaluation for nonlinearity in the association between predictors and outcomes. We considered a nominal p value of ≤ 0.05 as statistically significant and employed Bonferroni method for adjusting the p-values for multiple
Table 1 Baseline demographic, clinical and MRI. Demographics and clinical characteristics (N = 42) % Female Mean age in years ± SD Mean disease duration in months ± SD % White Mean education in years ± SD Median EDSS (range) Mean PASAT 3′ ± SD Mean T25FW (seconds) ± SD Mean 9-HPT (dominant hand; seconds) ± SD Mean MSFC score ± SD
71% 35.5 ± 9.6 7.5 ± 4.9 98% 15.5 ± 3.2 2.0 (0.0–5.5) 49.8 ± 10.6 5.1 ± 2.8 19.8 ± 4.3 0.010 ± 0.794
Brain Imaging Mean nBPV (cm3) ± SD Mean nGMV (cm3) ± SD Mean T2 lesion volume (cm3) ± SD Mean thalamic volume (cm3) ± SD Mean thalamic fraction ± SDa Mean cerebellar cortical volume (cm3) ± SD Mean cerebellar cortical fraction ± SDa Mean caudate volume (cm3) ± SD Mean caudate fraction ± SDa Mean putamen volume (cm3) ± SD Mean putamen fraction ± SDa Mean globus pallidus volume (cm3) ± SD Mean globus pallidus fraction ± SDa
1640 ± 118 908 ± 70.7 4.3 ± 5.6 7.5 ± 1.0 9.8 ± 0.8 110.5 ± 13.8 72.5 ± 6.6 3.7 ± 0.5 4.9 ± 0.5 5.4 ± 0.7 7.1 ± 0.9 1.2 ± 0.2 1.6 ± 0.2
EDSS: Expanded disability status scale; PASAT: Paced auditory serial addition test; T25FW: Timed 25-ft walk test; 9-HPT: 9-hole peg test; MSFC: Multiple sclerosis functional composite; nBPV: Normalized brain parenchymal volume; nGMV: Normalized grey matter volume. a Derived by dividing the structure volume by total intracranial volume; multiplied by 1000. The resulting numbers are unitless.
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Fig. 1. Linear prediction of change in the MRI measures over time based on the mixed-effects models (±95% confidence interval).
3.4. Cross-sectional association of baseline grey matter structures volumes and disability outcomes Estimated correlations were small and not statistically significant (p N 0.05) between baseline MRI variables and disability outcomes (data not shown). 3.5. Baseline grey matter structures volumes as predictors of longitudinal changes of disability outcomes Baseline thalamus, volume predicted subsequent change in the T25FW (p = 0.036) (Fig. 2). There was a trend for the association between baseline caudate volume and longitudinal change in T25FW (p = 0.084) Baseline thalamic volume predicted subsequent change in MSFC (p = 0.024). There was no statistically significant association between baseline grey matter volumes and EDSS. 3.6. Longitudinal changes in grey matter structures volumes as predictors of changes in disability outcomes There was no statistically significant or clinically meaningful association between longitudinal changes in the studied grey matter Table 2 Change in the MRI measures over time.
Thalamus fraction Cerebellar cortical fraction Caudate fraction Putamen fraction Pallidum fraction
% change in one year
95% CI
p-Value
−1.44 0.14 −2.78 −2.46 0.05
−2.3 to −0.57 −0.78–1.08 −3.5 to −2.05 −3.41 to −1.50 1.4–1.5
0.001 0.76 b0.001 b0.001 0.94
structures and within-subject changes in the clinical outcomes (data not shown). 4. Discussion In the current study, we demonstrated a clinically and statistically significant rate of atrophy in several sub-cortical grey matter nuclei despite interferon therapy. The annual rate of caudate and putamen atrophy in this cohort was particularly striking. Our results are in line with a longitudinal analysis of sub-cortical deep grey matter atrophy reporting mean decrease in caudate volume of 4 and 6.2% over 2 years in stable CIS and those CIS who converted to clinically definite MS respectively [15]. On the other hand, the rate of caudate atrophy in patients with a mean disease duration of 4 and 6 years (in the stable and progressive group, respectively) was 4.5 and 5.9% over 5 years [16]. This disparity might be explained by the difference in disease duration in these studies. It is also conceivable that the rate of atrophy in these 2 basal ganglia structures are higher at the beginning of the disease; as it was recently reported that putamen atrophy progresses in a degressive manner: the rate of atrophy is fastest during the first few years after the disease onset and slows down afterward [17] There is also a possibility that neurodegeneration is more severe in the current group of patients and they will reach the progressive stage of the disease faster. In contrast, the rate of atrophy in cerebellar cortex and pallidum was small and statistically non-significant in our study. The change in the relative contribution of direct grey matter inflammatory lesions versus neuronal loss due to dying back transected axons in different stages of the disease might explain these differences. As explained in the original report of the trial, we believe pseudoatrophy was not seen in this group of patients. Furthermore; pseudoatrophy is less likely to affect grey matter structure.
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Fig. 2. Change in the timed 25-ft walk T25FW over time (fitted values) according to patient's baseline thalamic volume. According to the model, patients with lowest baseline caudate volume had subsequent increase in their T25FW over time (A); while those with highest baseline caudate volume at baseline had a subsequent decrease in their T25FW over time (B).
B. Nourbakhsh et al. / Journal of the Neurological Sciences 366 (2016) 229–233
Walking is one of the most heavily affected functions in patients with MS and measures of gait impairment are one of the most informative components of any disability outcome measures [18]. The association between volume of basal ganglia structures and walking speed in older adults and in patients with Parkinson's disease has been reported [19,20]. A recent cross-sectional study of MS patients with longstanding disease reported correlation of pallidal and caudate volumes with the walking function [21]. Another cross-sectional study found an association between sub-cortical grey matter volume and T25FW [22]. In the current study, despite finding no cross-sectional correlation between grey matter volumes and clinical measures, baseline thalamic (and to some extent, caudate) volume predicted subsequent change in the T25FW. Patients with highest baseline volumes generally had no change or experienced improvement in the walking function, while patients with the lowest baseline volumes experienced worsening in the T25 FW. Basal ganglia circuits have important functions regulating posture and locomotion and higher density of neurons in these structures may protect MS patients from deterioration in walking function. The same was true for the association of baseline thalamic volume and change in the overall disability as measured by MSFC (and not EDSS). Although one study reported cross-sectional correlation between thalamic and caudate volumes and EDSS [23], another study found an association between grey matter atrophy with the MSFC, but not EDSS. This might be related to higher sensitivity of the MSFC to change early in the disease course [24]. The strengths of our study include frequent and systematic longitudinal measurement of clinical outcomes and MRI measures in a homogenous cohort of patients with very early MS in the setting of a clinical trial. We also used a statistical model that could take advantage of collected data at all time points, separating the effects of between- and within-subject changes over time and adjusted our analyses for multiple-hypothesis testing by a conservative method. This study also has several limitations. The limited sample size might have decreased the power to detect a small association between longitudinal change in the MRI and clinical measures. We also combined riluzole and placebo groups in our analyses. Unknown or unmeasured variables might have confounded the association between MRI and clinical measures. It was recently reported that the choice of the analysis method affects grey matter volume estimation [25]. We did not have measures of grey matter atrophy in the cord. We also did not have volumetric data from age-matched controls obtained from the same scanner. However, comparing with normal control data from a study with participants of relatively similar ages (mean age of 33.5 versus 35.5 years in our study) that used the same segmentation technique [26], there was a statistically significant difference between baseline thalamic volume of our participants and normal subjects in that study. Mean baseline thalamic volume in our study was 7.5 cm3 with standard deviation of 1.0, while the thalamic volume of 42 normal subjects in Calabrese et al. study [26] was 7.9 cm3 with standard deviation of 0.6 (two sided Student ttest p-value = 0.029). This is an indication of the possible presence of thalamic atrophy at baseline in our cohort. In conclusion, we found that the volume of some of the deep grey matter but not cortical structures affects the trajectory of change in the walking function and disability in patients with very early MS. These findings can guide the future research of prognostic markers of disability and possibly help with the design of neuroprotection trials in early disease. Study funding Bardia Nourbakhsh is a grantee of National MS Society. This research was conducted while B.N. was an American Brain Foundation and a Biogen Idec Postdoctoral Fellow. Amir-Hadi Maghzi was funded by the Multiple Sclerosis International Federation (www.msif.org) through a
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McDonald Fellowship. This research was performed as a research grant funded by the National MS Society (PI Waubant, RG3932-A-2).
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Mowry, et al., A randomized controlled phase II trial of riluzole in early multiple sclerosis, Ann. Clin. Transl. Neurol. 1 (5) (May 2014) 340–347. [11] J.F. Kurtzke, Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS), Neurology 33 (11) (Nov 1983) 1444–1452. [12] G.R. Cutter, M.L. Baier, R.A. Rudick, D.L. Cookfair, J.S. Fischer, J. Petkau, et al., Development of a multiple sclerosis functional composite as a clinical trial outcome measure, Brain J. Neurol. 122 (Pt 5) (May 1999) 871–882. [13] M. Reuter, N.J. Schmansky, H.D. Rosas, B. Fischl, Within-subject template estimation for unbiased longitudinal image analysis, NeuroImage 61 (4) (Jul 16 2012) 1402–1418. [14] E. Vittinghoff, D.V. Glidden, S.C. Shiboski, C.E. McCulloch, Regression Methods in Biostatistics [Internet], Springer US, Boston, MA, 2012 ([cited 2015 Aug 7]. (Statistics for Biology and Health). Available from: http://link.springer.com/10.1007/978-1-46141353-0). [15] R. 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Grabli, et al., MRI atrophy of the caudate nucleus and slower walking speed in the elderly, NeuroImage 60 (2) (Apr 2 2012) 871–878. [20] M.R. DeLong, T. Wichmann, Circuits and circuit disorders of the basal ganglia, Arch. Neurol. 64 (1) (Jan 1 2007) 20–24. [21] R.W. Motl, E.A. Hubbard, N. Sreekumar, N.C. Wetter, B.P. Sutton, L.A. Pilutti, et al., Pallidal and caudate volumes correlate with walking function in multiple sclerosis, J. Neurol. Sci. 354 (1–2) (Jul 15 2015) 33–36. [22] R.W. Motl, R. Zivadinov, N. Bergsland, R.H. Benedict, Thalamus volume and ambulation in multiple sclerosis: a cross-sectional study, Neurodegener. Dis. Manag. 6 (1) (Feb 2016) 23–29. [23] G. Tao, S. Datta, R. He, F. Nelson, J.S. Wolinsky, P.A. Narayana, Deep gray matter atrophy in multiple sclerosis: a tensor based morphometry, J. Neurol. Sci. 282 (1–2) (Jul 15 2009) 39–46. [24] M.D. Goldman, R.W. Motl, R.A. Rudick, Possible clinical outcome measures for clinical trials in patients with multiple sclerosis, Ther. Adv. Neurol. Disord. 3 (4) (Jul 2010) 229–239. [25] V. Popescu, M.M. Schoonheim, A. Versteeg, N. Chaturvedi, M. Jonker, R. Xavier de Menezes, et al., Grey matter atrophy in multiple sclerosis: clinical interpretation depends on choice of analysis method, PLoS ONE 11 (1) (2016) e0143942. [26] M. Calabrese, F. Rinaldi, I. Mattisi, V. Bernardi, A. Favaretto, P. Perini, et al., The predictive value of gray matter atrophy in clinically isolated syndromes, Neurology 77 (3) (Jul 19 2011) 257–263.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Subcortical grey matter volumes predict subsequent walking function in early multiple sclerosis Bardia Nourbakhsh a,⁎, Christina Azevedo c, Amir-Hadi Maghzi d, Rebecca Spain e, Daniel Pelletier c, Emmanuelle Waubant a,b a
Department of Neurology, University of California San Francisco, San Francisco, CA, United States Department of Pediatrics, University of California San Francisco, San Francisco, CA, United States Department of Neurology, University of Southern California, Los Angeles, CA, United States d Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA, United States e Department of Neurology, Oregon Health and Science University, Portland, OR, United States b c
a r t i c l e
i n f o
Article history: Received 13 January 2016 Received in revised form 4 April 2016 Accepted 28 April 2016 Available online 06 May 2016 Keywords: Multiple sclerosis Grey matter Ambulation Atrophy
a b s t r a c t Background: Atrophy of subcortical grey matter structures has been reported to be associated with clinical measures of disability in multiple sclerosis (MS) patients. It is not clear if the degree of tissue loss in patients with very early MS is associated with changes in disability measures. Objective: To study the association between subcortical grey matter structure volumes and clinical disability outcomes. Methods: Relapsing MS patients within 12 months of clinical onset were enrolled in a neuroprotection trial of riluzole versus placebo with up to 36 months of follow-up and serial brain MRI and clinical assessments. MRI metrics, including thalamic, putamen, caudate, pallidum and cerebellar cortical volume, were measured by an automated, custom-made FreeSurfer pipeline. Volumes were normalized for head size. Clinical measures included EDSS, MSFC scores and its components. Mixed model regression measured time trends and associations between imaging and clinical outcomes. Results: 42 patients with a mean follow-up of 30.6 months were analyzed in this study. There was a statistically significant decrease in thalamus, caudate and putamen volumes, but not cerebellar cortical and pallidum volumes during the follow-up period. Baseline thalamus, caudate and putamen volumes predicted subsequent changes in the timed 25-ft walk test (p = 0.036) and MSFC (p = 0.024). There was a trend for an association between baseline caudate volume and subsequent change in the timed 25-ft walk test (p = 0.084). No association between baseline imaging and subsequent EDSS changes were seen. Conclusion: Subcortical grey matter volumes at early stages of MS are associated with subsequent changes in disability measures. © 2016 Elsevier B.V. All rights reserved.
1. Introduction MS has been traditionally defined as an inflammatory, demyelinating disease affecting the white matter in the central nervous system (CNS); however, grey matter pathology is increasingly recognized to occur early and throughout the disease course, and is associated of irreversible disability [1]. Grey matter pathology can be evaluated by different methods, including studying grey matter lesions, volumetric changes or assessment ⁎ Corresponding author at: Department of Neurology, University of California San Francisco, 675 Nelson Rising Lane, Room 221F, San Francisco, CA, Box 3206, 94158, United States. E-mail address: [email protected] (B. Nourbakhsh).
http://dx.doi.org/10.1016/j.jns.2016.04.054 0022-510X/© 2016 Elsevier B.V. All rights reserved.
of diffusion, magnetization transfer or spectroscopic changes [2]. Atrophy of the grey matter structures in the brain and spinal cord is a surrogate for neuronal loss. It better correlates with or predicts reaching disability milestones as compared to the markers of white matter injury [3]. Grey matter atrophy has been reported in patients within earliest stages of MS. [4–7] It has also been shown to correlate with physical disability [6,8,9]. However; this association has been shown in crosssectional studies and in patients with long-standing disease. Studying neuronal loss and its relevance to clinical measures of performance and disability will help design future neuroprotection trials in early MS which might be an ideal time for intervention. Our aim was to study the cross-sectional and longitudinal associations of brain deep grey matter structures and disability outcomes as measured by Expanded Disability Status Scale (EDSS), timed 25-ft walk (T25FW), 9-hole peg
230
B. Nourbakhsh et al. / Journal of the Neurological Sciences 366 (2016) 229–233
test (9HPT) and paced auditory serial addition test (PASAT) in very early MS. 2. Methods We analyzed data from a 2-year randomized double blind, placebo controlled trial of riluzole vs placebo. Details of the study, inclusion and exclusion criteria and the report of primary and secondary endpoints have been previously reported [10]. Relapsing-remitting and clinically isolated syndrome (CIS) patients within 12 months of symptom onset were offered participation in the trial. The first half of patients who completed the 24-month core study were offered continuation for another 12 months (mean follow-up of 30.6 months). Three months after randomizations, all patients started weekly interferon beta-1a. The study was approved by the institutional review boards of the participating centers (UCSF, OHSU). Informed consent was obtained from all participants before enrollment. Because riluzole, as compared to placebo did not show any significant effect on the primary and secondary outcomes of the study (including no effect on the clinical measures or grey matter structures), we combined both treatment groups for the analyses of the associations.
hypothesis testing. All analyses were conducted in Stata Version 13.1 (Stata Corp, College Station TX).
3. Results 3.1. Patient's characteristics Baseline demographic, clinical and radiographic characteristics of patients who were recruited in the study are shown in Table 1. Forty two of 43 patients enrolled in the clinical trial contributed to the longitudinal assessment of the association between MRI and clinical measures. MRI images of one patient could not be processed by the FreeSurfer. Only 5 patients (2 in the riluzole group and 3 in the placebo group) switched to another disease-modifying medication during the study, as treating physicians determined there was clinical or radiological disease activity on intramuscular weekly IFN-beta 1a. Relapse rate was 0.22 per year in the placebo and 0.15 in the riluzole group (p = 0.27).
3.2. Longitudinal changes in grey matter structures
EDSS and MSFC [including PASAT3′] were used to monitor neurological changes [11,12]. Patients had screening and baseline MSFC to allow for practice effects. The screening MSFC was not used in the analyses.
During the study, there was a statistically significant decrease in the volume of thalamus (1.4% per year, 95%CI: 0.6%–2.3%, p = 0.001), caudate (2.8% per year, 95%CI: 2.1%–3.5%, p b 0.001) and putamen (2.5% per year, 95CI: 1.5%–3.4%, p b 0.001), but not cerebellar cortical and pallidum volumes (Fig. 1 and Table 2).
2.2. MRI acquisition and post-processing
3.3. Longitudinal changes in the clinical measures
Details of the MRI protocol have been described previously [10]. Brain MRIs were performed on 3.0 Tesla MRI scanners equipped with an 8-channel phased array coil (General Electric, Milwaukee, WI) at baseline and months 6, 12, 18, 24 and for half of patients, at month 36. The standardized study protocol included a 3D, T1-weighted, volumetric, 1 mm-isotropic inversion recovery spoiled gradient-echo sequence (3D-IRSPGR, 1 × 1 × 1 mm3, 180 slices), which was used for all brain volume measurements, as well as a 2D multislice dual spin echo sequence (proton density and T2-weighted, 1 mm × 1 mm × 3 mm, no gaps). T1 lesion masks were used to inpaint 3D-IRSPGR images prior to submission to FreeSurfer [13] to avoid voxel misclassification errors. Anatomic segmentation was performed using FreeSurfer's longitudinal processing stream (v5.3; probabilistic atlas for subcortical segmentation) [13]. FreeSurfer's output was reviewed, manually corrected and reran as needed by an experienced MRI postprocessor. Deep grey and cerebellar cortical volumes were extracted directly from FreeSurfer's output and normalized for head size using the estimated total intracranial volume, also taken from FreeSurfer. The resulting values are unitless.
During the study, there was a statistically significant worsening in EDSS (0.18 point/year, 95%CI: 0.07–0.28, p = 0.005). There was no statistically significant longitudinal change in other clinical measures over time.
2.1. Clinical measurements
2.3. Statistical analysis Descriptive statistics for patient characteristics were presented either as percentages (%) or using mean ± standard deviation (SD). Spearman correlation was used to assess the cross-sectional association of clinical and imaging outcomes at baseline. Mixed effects regression models were used to account for the longitudinal nature of the data. To separate the between-participant from the within-participant effects, the baseline values and change from the baseline values of imaging markers were entered in the model as predictors [14]. We performed adequate model checking, including evaluation for nonlinearity in the association between predictors and outcomes. We considered a nominal p value of ≤ 0.05 as statistically significant and employed Bonferroni method for adjusting the p-values for multiple
Table 1 Baseline demographic, clinical and MRI. Demographics and clinical characteristics (N = 42) % Female Mean age in years ± SD Mean disease duration in months ± SD % White Mean education in years ± SD Median EDSS (range) Mean PASAT 3′ ± SD Mean T25FW (seconds) ± SD Mean 9-HPT (dominant hand; seconds) ± SD Mean MSFC score ± SD
71% 35.5 ± 9.6 7.5 ± 4.9 98% 15.5 ± 3.2 2.0 (0.0–5.5) 49.8 ± 10.6 5.1 ± 2.8 19.8 ± 4.3 0.010 ± 0.794
Brain Imaging Mean nBPV (cm3) ± SD Mean nGMV (cm3) ± SD Mean T2 lesion volume (cm3) ± SD Mean thalamic volume (cm3) ± SD Mean thalamic fraction ± SDa Mean cerebellar cortical volume (cm3) ± SD Mean cerebellar cortical fraction ± SDa Mean caudate volume (cm3) ± SD Mean caudate fraction ± SDa Mean putamen volume (cm3) ± SD Mean putamen fraction ± SDa Mean globus pallidus volume (cm3) ± SD Mean globus pallidus fraction ± SDa
1640 ± 118 908 ± 70.7 4.3 ± 5.6 7.5 ± 1.0 9.8 ± 0.8 110.5 ± 13.8 72.5 ± 6.6 3.7 ± 0.5 4.9 ± 0.5 5.4 ± 0.7 7.1 ± 0.9 1.2 ± 0.2 1.6 ± 0.2
EDSS: Expanded disability status scale; PASAT: Paced auditory serial addition test; T25FW: Timed 25-ft walk test; 9-HPT: 9-hole peg test; MSFC: Multiple sclerosis functional composite; nBPV: Normalized brain parenchymal volume; nGMV: Normalized grey matter volume. a Derived by dividing the structure volume by total intracranial volume; multiplied by 1000. The resulting numbers are unitless.
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Fig. 1. Linear prediction of change in the MRI measures over time based on the mixed-effects models (±95% confidence interval).
3.4. Cross-sectional association of baseline grey matter structures volumes and disability outcomes Estimated correlations were small and not statistically significant (p N 0.05) between baseline MRI variables and disability outcomes (data not shown). 3.5. Baseline grey matter structures volumes as predictors of longitudinal changes of disability outcomes Baseline thalamus, volume predicted subsequent change in the T25FW (p = 0.036) (Fig. 2). There was a trend for the association between baseline caudate volume and longitudinal change in T25FW (p = 0.084) Baseline thalamic volume predicted subsequent change in MSFC (p = 0.024). There was no statistically significant association between baseline grey matter volumes and EDSS. 3.6. Longitudinal changes in grey matter structures volumes as predictors of changes in disability outcomes There was no statistically significant or clinically meaningful association between longitudinal changes in the studied grey matter Table 2 Change in the MRI measures over time.
Thalamus fraction Cerebellar cortical fraction Caudate fraction Putamen fraction Pallidum fraction
% change in one year
95% CI
p-Value
−1.44 0.14 −2.78 −2.46 0.05
−2.3 to −0.57 −0.78–1.08 −3.5 to −2.05 −3.41 to −1.50 1.4–1.5
0.001 0.76 b0.001 b0.001 0.94
structures and within-subject changes in the clinical outcomes (data not shown). 4. Discussion In the current study, we demonstrated a clinically and statistically significant rate of atrophy in several sub-cortical grey matter nuclei despite interferon therapy. The annual rate of caudate and putamen atrophy in this cohort was particularly striking. Our results are in line with a longitudinal analysis of sub-cortical deep grey matter atrophy reporting mean decrease in caudate volume of 4 and 6.2% over 2 years in stable CIS and those CIS who converted to clinically definite MS respectively [15]. On the other hand, the rate of caudate atrophy in patients with a mean disease duration of 4 and 6 years (in the stable and progressive group, respectively) was 4.5 and 5.9% over 5 years [16]. This disparity might be explained by the difference in disease duration in these studies. It is also conceivable that the rate of atrophy in these 2 basal ganglia structures are higher at the beginning of the disease; as it was recently reported that putamen atrophy progresses in a degressive manner: the rate of atrophy is fastest during the first few years after the disease onset and slows down afterward [17] There is also a possibility that neurodegeneration is more severe in the current group of patients and they will reach the progressive stage of the disease faster. In contrast, the rate of atrophy in cerebellar cortex and pallidum was small and statistically non-significant in our study. The change in the relative contribution of direct grey matter inflammatory lesions versus neuronal loss due to dying back transected axons in different stages of the disease might explain these differences. As explained in the original report of the trial, we believe pseudoatrophy was not seen in this group of patients. Furthermore; pseudoatrophy is less likely to affect grey matter structure.
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Fig. 2. Change in the timed 25-ft walk T25FW over time (fitted values) according to patient's baseline thalamic volume. According to the model, patients with lowest baseline caudate volume had subsequent increase in their T25FW over time (A); while those with highest baseline caudate volume at baseline had a subsequent decrease in their T25FW over time (B).
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Walking is one of the most heavily affected functions in patients with MS and measures of gait impairment are one of the most informative components of any disability outcome measures [18]. The association between volume of basal ganglia structures and walking speed in older adults and in patients with Parkinson's disease has been reported [19,20]. A recent cross-sectional study of MS patients with longstanding disease reported correlation of pallidal and caudate volumes with the walking function [21]. Another cross-sectional study found an association between sub-cortical grey matter volume and T25FW [22]. In the current study, despite finding no cross-sectional correlation between grey matter volumes and clinical measures, baseline thalamic (and to some extent, caudate) volume predicted subsequent change in the T25FW. Patients with highest baseline volumes generally had no change or experienced improvement in the walking function, while patients with the lowest baseline volumes experienced worsening in the T25 FW. Basal ganglia circuits have important functions regulating posture and locomotion and higher density of neurons in these structures may protect MS patients from deterioration in walking function. The same was true for the association of baseline thalamic volume and change in the overall disability as measured by MSFC (and not EDSS). Although one study reported cross-sectional correlation between thalamic and caudate volumes and EDSS [23], another study found an association between grey matter atrophy with the MSFC, but not EDSS. This might be related to higher sensitivity of the MSFC to change early in the disease course [24]. The strengths of our study include frequent and systematic longitudinal measurement of clinical outcomes and MRI measures in a homogenous cohort of patients with very early MS in the setting of a clinical trial. We also used a statistical model that could take advantage of collected data at all time points, separating the effects of between- and within-subject changes over time and adjusted our analyses for multiple-hypothesis testing by a conservative method. This study also has several limitations. The limited sample size might have decreased the power to detect a small association between longitudinal change in the MRI and clinical measures. We also combined riluzole and placebo groups in our analyses. Unknown or unmeasured variables might have confounded the association between MRI and clinical measures. It was recently reported that the choice of the analysis method affects grey matter volume estimation [25]. We did not have measures of grey matter atrophy in the cord. We also did not have volumetric data from age-matched controls obtained from the same scanner. However, comparing with normal control data from a study with participants of relatively similar ages (mean age of 33.5 versus 35.5 years in our study) that used the same segmentation technique [26], there was a statistically significant difference between baseline thalamic volume of our participants and normal subjects in that study. Mean baseline thalamic volume in our study was 7.5 cm3 with standard deviation of 1.0, while the thalamic volume of 42 normal subjects in Calabrese et al. study [26] was 7.9 cm3 with standard deviation of 0.6 (two sided Student ttest p-value = 0.029). This is an indication of the possible presence of thalamic atrophy at baseline in our cohort. In conclusion, we found that the volume of some of the deep grey matter but not cortical structures affects the trajectory of change in the walking function and disability in patients with very early MS. These findings can guide the future research of prognostic markers of disability and possibly help with the design of neuroprotection trials in early disease. Study funding Bardia Nourbakhsh is a grantee of National MS Society. This research was conducted while B.N. was an American Brain Foundation and a Biogen Idec Postdoctoral Fellow. Amir-Hadi Maghzi was funded by the Multiple Sclerosis International Federation (www.msif.org) through a
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McDonald Fellowship. This research was performed as a research grant funded by the National MS Society (PI Waubant, RG3932-A-2).
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