Magnetisation transfer, diffusion and g-ratio measures of demyelination and neurodegeneration in early relapsing-remitting multiple sclerosis: a longitudinal microstructural MRI study.

Introduction Relapsing-remitting multiple sclerosis (RRMS) is a chronic, immune-mediated neurodegenerative disease with a highly heterogeneous disease course. Conventional MRI is widely used for diagnosis and disease monitoring but provides limited specific information on MS-related damage. Improved in vivo biomarkers, which are sensitive to subtle changes in RRMS pathology, are needed for early patient stratification and evaluation of neuroprotective and disease modifying therapies (DMTs). Quantitative MRI methods, such as myelin-sensitive magnetisation transfer ratio (MTR) or saturation (MTsat) imaging, axon-sensitive diffusion Neurite Orientation Dispersion and Density Imaging (NODDI) and derived aggregate measures (e.g. MR g-ratio) may provide more specific indices of microstructural damage in RRMS. The aim of this study was to determine the sensitivity of microstructural MRI markers (magnetisation transfer ratio [MTR], MTsat, NODDI isotropic/intraneurite volume fractions, and g-ratio) to myelin and axonal damage in white matter lesions (WML) and normal appearing white matter (NAWM) and their longitudinal change in early RRMS. Methods Seventy-seven people were recruited to an extended MRI sub-study of Future-MS, a longitudinal study of people with recently diagnosed RRMS. MR imaging and clinical assessment were performed at baseline, prior to initiation of any DMTs, and one year later. Twelve healthy volunteers additionally received the same MR protocol, repeated within two weeks, to determine test-retest agreement. Approval was granted from the local Research Ethics committee and participants provided written informed consent. A 3T MR imaging protocol included structural 3D T1-weighted and T2-weighted FLAIR sequences, magnetisation transfer acquisition (comprising two proton density images with and without a saturation pulse, plus a T1-weighted image), and multishell diffusion-weighted 2D echo-planar imaging. MTR and MTsat were calculated from magnetisation transfer data, and NODDI isotropic and intraneurite volume fractions (ISOVF and ICVF, respectively) from diffusion-weighted data. The g-ratio was calculated from MTsat and NODDI data. WML and NAWM tissue masks were segmented from structural images. Tissue contrast for MTsat and MTR was compared with paired t-tests. Voxelwise distributions of microstructural metrics were examined graphically. Longitudinal change in MR-derived measures in NAWM and WML was assessed with paired t-tests (=0.05) with follow-up linear mixed models, where significant, to control for confounding factors, with False Discovery Rate (FDR) correction for multiple comparisons. Results Complete longitudinal data for MTsat and MTR was available for sixty-two patients, and sixty patients for NODDI and g-ratio. Contrast between NAWM and WML was greater for MTsat than MTR (mean difference = 16.9 [95% CI 15.96 to 17.89], paired t-test: t(74)=34.97, p<0.001). Voxelwise histograms showed a negative linear dependence on the approximation of T1 recovery (T1app) for MTsat, but not MTR, in NAWM and a non-linear association in WML. Simulations and voxelwise histograms demonstrated that g-ratio is inversely related to MTsat and NODDI ISOVF but positively related to NODDI ICVF. MTsat and NODDI ICVF were not associated in control white matter or NAWM but broadly co-correlated in WML. In NAWM, g-ratio and NODDI ICVF increased significantly over one year (mean difference = 0.004 [95% CI 0.001 to 0.007] and 0.002 [0.0002 to 0.004], paired t-test: t(59) = 2.60 and 2.29, p = 0.012 and 0.025 respectively) and MTsat decreased (mean difference = -0.032 [95% CI -0.061 to -0.003], t(61) = -2.19, p = 0.033). Neither MTR nor NODDI ISOVF changed significantly over one year (p=0.94 and p=0.67). After accounting for covariates and adjustment for multiple comparisons, the longitudinal increase in g-ratio and in NODDI ICVF remained significant (linear mixed model: adjusted mean difference = 0.007 and 0.004, t(75.9) = 3.08 and t(90.6) = 3.51, FDR-corrected p = 0.029 and 0.007). The decrease in MTsat did not survive correction for multiple comparisons (FDR-corrected p = 0.11). In WML, paired t-tests revealed increases in MTsat, MTR, NODDI ISOVF and ICVF over one year, but no change in g-ratio. Follow-up linear mixed models, however, showed that only changes in MTsat and NODDI metrics were significant after correction for confounding factors and multiple comparisons. Longitudinal change did not, however, generally exceed healthy control test-retest limits of agreement except for NODDI ISOVF and ICVF in WML. Discussion Higher tissue contrast for MTsat compared with MTR suggests MTsat is more sensitive to demyelination than widely used MTR. Comparisons of microstructural MRI data distributions indicate that MTsat is better suited to measurement of subtle myelin loss in NAWM compared with MTR. NAWM data distributions suggest that MTsat and NODDI ICVF provide independent measures of myelin and axonal integrity. Simulations show that g-ratio increases as MTsat decreases, but changes in NODDI parameters may complicate interpretation of longitudinal change in g-ratio. The observed increase in g-ratio and MTsat in NAWM is indicative of subtle myelin loss in patients with early RRMS. The unexpected increase observed in NODDI ICVF suggests axonal swelling and/or axonal repair, and may confound g-ratio. Increases in MTsat and NODDI ICVF in WML are also suggestive of tissue repair. WML g-ratio did not change over time, likely due to competing effects of longitudinal MTsat and NODDI changes. Despite significant groupwise effects, detection of longitudinal change at an individual level may be limited by technique test-retest agreement in early RRMS. Conclusion MTsat and g-ratio are promising in vivo biomarkers of myelin integrity in early RRMS, and appear more sensitive than MTR for detecting subtle demyelination. Our findings suggest that MTsat and g-ratio measures may be most sensitive for detecting subtle change in NAWM, as compared with more severely damaged WML where T1 effects and neuroaxonal damage predominate. Complex dependence of g-ratio on NODDI parameters illustrates the limitation of interpreting such aggregate measures in isolation, and independent consideration of myelin-sensitive and axonal neuroimaging markers may ultimately be more informative for longitudinal tracking of neuropathology in RRMS. Technique reproducibility currently limits evaluation in individual patients, and future research is warranted to validate diffusion and MT models across heterogeneous microstructural damage found in MS, for translation into clinical practice and trial platforms.