MR spectroscopy (MRS) and magnetisation transfer imaging (MTI), lesion load and clinical scores in early relapsing remitting multiple sclerosis: a combined cross-sectional and longitudinal study
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- Bellmann-Strobl, J., Stiepani, H., Wuerfel, J. et al. Eur Radiol (2009) 19: 2066. doi:10.1007/s00330-009-1364-z
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The purpose of this study was to correlate magnetic resonance imaging (MRI)-based lesion load assessment with clinical disability in early relapsing remitting multiple sclerosis (RRMS). Seventeen untreated patients (ten women, seven men; mean age 33.0 ± 7.9 years) with the initial diagnosis of RRMS were included for cross-sectional as well as longitudinal (24 months) clinical and MRI-based assessment in comparison with age-matched healthy controls. Conventional MR sequences, MR spectroscopy (MRS) and magnetisation transfer imaging (MTI) were performed at 1.5 T. Lesion number and volume, MRS and MTI measurements for lesions and normal appearing white matter (NAWM) were correlated to clinical scores [Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC)] for monitoring disease course after treatment initiation (interferon β-1a). MTI and MRS detected changes [magnetisation transfer ratio (MTR), N-acetylaspartate (NAA)/creatine ratio] in NAWM over time. EDSS and lesional MTR increases correlated throughout the disease course. Average MTR of NAWM raised during the study (p < 0.05) and correlated to the MSFC score (r = 0.476, p < 0.001). At study termination, NAA/creatine ratio of NAWM correlated to the MSFC score (p < 0.05). MTI and MRS were useful for initial disease assessment in NAWM. MTI and MRS correlated with clinical scores, indicating potential for monitoring the disease course and gaining new insights into treatment-related effects.
KeywordsMultiple sclerosisMR spectroscopyMagnetisation transfer imagingWhite matter
Multiple sclerosis (MS) is the most common demyelinating disease worldwide and the most common neurological disorder within the northern hemisphere in young adults, showing a prevalence of 0.1% . Different MS disease courses as well as varying therapeutic regimens require early and subtle assessment of acute and chronic parenchymal inflammation.
Magnetic resonance imaging (MRI) has proven to be the imaging technique of choice in establishing the diagnosis as well as for disease monitoring. MRI plays an important role in MS diagnostics, as underlined by the so-called revised McDonald criteria of the International Panel on the Diagnosis of Multiple Sclerosis .
Advanced MRI techniques such as MR spectroscopy (MRS) and magnetisation transfer imaging (MTI) have shown the unprecedented ability to depict and quantify changes in lesions and normal appearing white matter (NAWM) over time [3–5]. MRS provides metabolic information and indicates neuronal loss by decrease of marker molecule concentrations such as N-acetylaspartate (NAA) , whereas MTI detects alterations in macromolecular components, e.g. of myelin , which cannot be depicted by conventional MRI.
This study aimed at evaluating the suitability of MRS and MTI in monitoring neuroinflammatory parenchymal brain damage in correlation with conventional MRI as well as different clinical disability scores at the time of initial diagnosis and throughout the disease course after initiating interferon β (IFN β) therapy in patients with early relapsing remitting multiple sclerosis (RRMS). Only a few studies have been carried out with mostly small sample sizes and lack of placebo control using either MRS or MTI for evaluating IFN-β treatment effects. Whether or not MRS is a suitable technique for therapy monitoring could not be clarified by these studies [8–11]. In a single small trial on RRMS patients treated with interferon β–1b (IFN β–1b), magnetisation transfer ratio (MTR) was not altered during a period of 6 months . However, lesional MTR was reported to evolve at a faster rate during IFN therapy and may be a surrogate measure of resolving demyelination . To our knowledge, this is the first longitudinal study combining MRS and MTI to investigate RRMS patients treated with IFN β-1a).
Materials and methods
Diagnosis of MS according to the McDonald criteria  at the time of study inclusion
No corticoidsteroid and/or immunmodulatory treatment throughout the preceding 6 months
No clinical signs of an acute inflammatory relapse
The patients in the study group were assessed clinically and by MRI at the time of initial MS diagnosis (i.e. the time of study inclusion) as well as at monthly intervals for 1 year and a single follow-up at 24 months.
The control group consisted of 17 healthy subjects (ten women, seven men; mean age 30.5 ± 7.0 years, range 20–45 years) with no clinical signs nor a history of neurological disorders. None of the participants had been treated with corticosteroids and/or other immunomodulatory drugs.
The study was approved by the local ethics committee. All subjects gave their written informed consent before inclusion in the study.
All MRI studies were performed using a 1.5-Tesla system (Magnetom Vision plus, Siemens Medical Solutions, Erlangen, Germany).
MR sequence parameters
Number of slices
Slice thickness (mm)
Distance factor (mm)
256 × 192
256 × 192
512 × 192
256 × 192
8 × 8
Lesion count and volumes were quantified based on T2-weighted double-echo sequences. These sequences were also applied for the segmentation of normal brain matter, NAWM and cerebrospinal fluid (CSF). The T1-weighted spin-echo sequence was used for determining the volume of so-called “black holes", corresponding to T1-hypointense, post-inflammatory lesions. As based on the available literature, a significant correlation between clinical scores and lesion enhancement could not be expected and, due to the tight time-line of re-scans, the decision was made not to apply contrast-enhanced measurements.
MTI measurements allowed for determining the average transfer ratio of lesions and WM. An off-resonance radiofrequency pulse with 1.5 kHz distance to the Larmor frequency of hydrogen, 16.4 ms duration, 850° flip angle and 768 Hz bandwidth was chosen. Measurements were programmed in a way that a gradient-echo sequence without off-resonance pulses was immediately followed by the one using off-resonance pulses.
Subsequently, the following parameters were determined in an automated manner: lesion count and volumes of T2-weighted studies, average lesion MTR and average MTR of whole brain WM. Altogether, 189 lesions were analysed throughout the study period. Average time consumption per session post-processing amounted to 120 min.
Each patient was assessed clinically on the MRI study day by an experienced neurologist; this included the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Functional Composite (MSFC). EDSS quantifies disability in MS with respect to eight functional systems by allowing the neurologist to assign a Functional System Score (FSS) in each of these. EDSS steps 0 to 3.5 refer to patients with MS who are fully ambulatory, EDSS steps 4.0 to 9.5 are defined by the impairment to movement.
The EDSS test was supplemented by the Multiple Sclerosis Functional Composite (MSFC) test as this is able to monitor subtle changes in the disease course as well as to reflect cognitive impairment induced by MS [15, 16]. MSFC is a multidimensional clinical outcome measure that includes quantitative tests of leg function/ambulation [Timed 25-Foot Walk (TWT)], arm function [9-Hole Peg Test (9-HPT)], and cognitive function [Paced Auditory Serial Addition Test (PASAT)]. From these three tests, so-called z-scores are derived representing the differences between the patients performance compared with the cohort investigated.
Ten out of 17 patients completed the study period of 24 months. They were treated with either IFN β-1a 22 μg s.c. (n = 7), IFN β-1a 30 μg i.m. (n = 1), glatiramer acetate (n = 1) or remained untreated (n = 1).
After a baseline scan, therapy start was initiated by applying IFN β-1a 22 μg s.c. three times per week, by injecting IFN β-1a 30 µg once per week or by daily injections of glatiramer acetate.
Statistical evaluation as well as diagram generation was achieved by using SPSS 11.5 (SPSS, Chicago, Ill., USA). In order to assess cross-sectional data differences at initial diagnosis the Mann-Whitney U-test as well as the Spearman’s rank correlation coefficient (rs) were used. For longitudinal data evaluation, the non-parametric Friedman test and the Wilcoxon signed-rank test for repeated measurements on a single sample were used as well as the Spearman’s rank correlation coefficient.
Seventeen patients with RRMS were included into the study, ten of which could be followed up to 24 months (longitudinal study) subsequently. Seven patients terminated their study participation prematurely for personal reasons, such as relocation etc.
One out of ten patients in the longitudinal study part was excluded from data assessment because of disease conversion from relapsing-remitting to a secondary progressive course at 15 months.
Another three of the remaining nine patients in the 2-year follow-up had to be excluded from data analysis because of differences in medication; one of these three patients remained untreated and was also followed also over a period of 24 months.
This added up to a group of six patients treated with IFN β-1a 22 μg and monitored in monthly intervals for 1 year with a follow-up after 24 months.
MRI studies of all control subjects remained without abnormal findings at any time-point throughout the study period. For whole brain NAWM in the control group, a mean MTR of 41.3% was determined.
All patients showed WM lesions on T2-weighted images with an average of 14.8 ± 11.8 lesions, 3.0 ± 3.9 ml in T2-weighted lesion load and 0.8 ± 1.3 ml in T1-weighted hypointense lesion volumes.
At the time of initial diagnosis, a broad range with respect to lesion count and volumes in T2- and T1-weighted studies was noted.
T2- and T1-weighted lesion volumes were correlated (rs = 0.871, p < 0.001). Lesion load and neurological scores (EDSS, MSFC) did not cohere significantly, whereas the z-score of 9-HPT, a MFSC subtest, did correlate with the T1-weighted lesion volumes (rs = -0.636, p < 0.05). Lesion count and volumes did not correlate with MRS nor MTI measurements.
MRS and MTI
Compared with healthy controls (NAA/Cr 2.07; Cho/Cr ratio 0.91), the NAA/Cr ratio in the periventricular WM and callosal body of patients was decreased to 1.89 ± 0.15 (p < 0.001) and the average Cho/Cr ratio was increased to 1.05 ± 0.09 (p < 0.001).
The average MTR of NAWM amounted to 38.51 ± 1.05%, average lesion MTR to 34.45 ± 3.09%. These results differed significantly from those of healthy controls (MTR 41.30 ± 0.74%; p < 0.001).
A statistical trend was observed towards a correlation between MSFC and MRS (NAA/Cr ratio) (rs = 0.439, p < 0.058). A correlation was noted between 9-HPT, a subtest of MSFC, and MRS (Cho/Cr ratio) (rs = 0.675, p < 0.01).
EDSS and MSFC scores did not correlate with MTI study results nor with conventional MRI data.
Mean NAA/Cr ratio and MTR values (%) in NAWM at baseline, 12 and 24 months after treatment initialisation (*p < 0.05)
NAA/Cr (VOI NAWM)
1.93 ± 0.06
2.04 ± 0.06
2.06 ± 0.05*
2.17 ± 0.07
2.12 ± 0.06
2.15 ± 0.05
MTR (%) (whole-brain NAWM)
39.33 ± 0.67
40.76 ± 0.94
40.97 ± 1.09*
41.57 ± 0.80
41.58 ± 0.61
41.89 ± 0.71
Neither lesion counts nor lesion volumes changed significantly throughout the study period.
MRS and MTI
EDSS values decreased (p < 0.05) and MSFC figures increased (p < 0.05) during the follow-up period of 24 months in the patient group.
EDSS correlated with MTR in NAWM (p < 0.001) as well as within lesions (p < 0.05).
MSFC correlated with MTR of NAWM (p < 0.001) as well as with the T2-weighted lesion load (p < 0.01). At 24 months MSFC correlated with NAA/Cr ratio (p < 0.05), as well as did 9-HPT at 2, 12 and 24 months (p < 0.05). EDSS showed a relation to the Chol/Cr ratio at 24 months (p < 0.05). EDSS and MSFC scores did not correlate with conventional MRI data at any time-point.
Whereas the MTR reflects important aspects of macromolecular changes during de- and remyelination in MS , MRS has proven useful in assessing neuronal damage, i.e. axonal loss, as NAA is exclusively localised in the neuronal compartment .
Recent surveys underline the potential of MTI and MRS in elucidating widespread tissue damage in NAWM at early MS stages, monitoring various aspects of inflammation in MS, characterising new, stable and resolved lesions, predicting the individual clinical course and monitoring treatment-related effects on myelin repair and neuroprotection [18, 21–23].
These results strongly support the use of MTI and MRS in clinical MS studies.
In our investigation, MR spectroscopy revealed significant changes in patients’ NAA/Cr as well as in Cho/Cr ratios of the periventricular WM and callosal body in comparison to healthy controls, most probably reflecting different pathophysiological mechanisms such as axonal damage (NAA/Cr ratio) , and inflammatory cell membrane turnover (Cho/Cr) . These results did not correlate with conventional lesion counts and volumes assessed on T1-/T2-weighted MR studies, indicating that advanced and conventional MR techniques, although both effective in differentiating patients and controls in our study, deliver different qualities of information in MS . Dissociation between MRS findings indicative of axonal loss and T2 lesion volumes at early stages of the disease have also been encountered in other studies .
Diffusely increased Cho levels in NAWM of RRMS patients  as well as decreased NAA concentrations in patients with clinically isolated syndromes (CIS) suggestive of MS have been described , presumably representing inflammatory changes and significant axonal damage early in the disease course. MR spectroscopy, i.e. NAA levels, might be a prognostic marker for CIS patients with a higher risk of conversion into definite MS, as signs of early axonal damage at baseline was more prominent in this group of patients .
Neither lesion counts nor lesion volumes changed significantly throughout the study period, whereas MRS depicted a predominantly continuous and significant increase in WM NAA/Cr ratio. This is in line with two MRS studies of IFN β treatment in RRMS [9, 11]; however, similar studies by Pascual-Lozano et al. , and Sarchielli et al.  could not confirm these results. There is growing evidence that in MS inflammation is not only limited to lesions but also a diffuse, chronic low-level process affecting the entire brain. Therefore, the rise of NAA/Cr is discussed to be not only a sign of axonal repair but also the expression of resolving axonal dysfunction due to the anti-inflammatory effects of IFN β .
These findings are supported by clinical studies which implicated that IFN β may prevent and potentially reverse axonal injury .
Correlations between MRS findings and clinical scores have been reported in both cross-sectional and longitudinal studies . In the cross-sectional part of our study, a statistical trend was observed towards a correlation between NAA/Cr ratio and MSFC (rs = 0.439, p < 0.058), in accordance with the findings reported by Adalsteinsson et al. , as well as well as a significant correlation between Cho/Cr ratio and 9-HPT, a subtest of MSFC (p < 0.01). Also in the course of 24 months MSFC correlated with NAA/Cr (p < 0.05). As there were no correlations between changes in conventional MRI parameters (T1 and T2 lesion count and volume) and disability, MRS seems to be a potential surrogate marker of disease progression.
Thus, advanced MRI techniques may be helpful in therapeutic decision making, e.g. with respect to early initiation of treatment with IFN β-1a as well as IFN β-1b, both of which were shown to be beneficial in counteracting disability development [35–37].
MTI proved suitable for detecting macromolecular tissue damage in the WM of MS patients without lesional evidence in conventional MR studies . Chen et al.  were able to validate the MTR changes in an MS lesion indicative of remyelination and demyelination by histopathological studies.
MTR changes in the NAWM of patients that correlated with clinical decline assessed by EDSS, MSFC and additional neuropsychological tests have recently been reported by other authors [25, 39, 40]. In our study, such correlations were only delineated during follow-up, not in the cross-sectional study part. The early stage of MS as well as a heterogeneous lesion distribution within our cross-sectional patient cohort might at least partially account for these differences. In addition, clinical alterations with respect to attention, information processing speed, memory, inhibition and conceptualisation which occur during early stages of RRMS  may not be addressed sensitively enough by the standard MS disability scores applied in this study.
The MTI findings of a continuous and significant MTR increase in NAWM support the assumption of brain parenchymal recovery. MTI provided the only imaging parameter (MTR) that correlated with the improvements seen in both clinical scores. Other studies report no change of MTR during treatment with IFN β . However, this discrepancy could be due to the fact that their patients suffered from more severe disease state with high frequency of enhancing lesions. In addition, in our data the NAA/Cr ratio as a second independently derived parameter also showed an increase, implicating a restoration of axonal integrity.
Yet, in studies of untreated MS patients, the MTR of NAWM showed a slowly progressive decrease that started at disease onset—as we observed in the untreated patient of our investigation—and accelerated rapidly in focal areas just prior to lesion appearance on conventional MRI, probably reflecting different disease courses .
Limitations of our study are the relatively small number of patients fulfilling the 2-year longitudinal study. Additionally, grey matter (GM) changes have not been addressed in our study, although there is growing evidence for the concept that MS comprises a neurodegenerative component, with different patterns of atrophy evolution in GM and WM tissue compartments . MT MRI assessment in GM has been shown to detect subtle brain tissue changes that are associated with mild clinical impairment as well as short-term disability accumulation in patients with RRMS [43, 44]. In addition, our data do not permit a statistically meaningful comparison between treated and untreated patients (only one patient remained untreated); yet, it may be speculated that the comparable MRS and MTR figures of the untreated patient and the healthy control subjects for the longest part of our study period suggest interindividual differences in susceptibility to acute inflammatory episodes in MS patients.
Our results suggest that advanced MR imaging techniques (MTI, MRS) perform better than conventional MRI in terms of detecting early parenchymal damage as well as reflecting the patients’ clinical status in relapsing remitting multiple sclerosis. Further studies with larger cohorts of patients are necessary to clarify the significance of these methods in monitoring therapeutic efficacy.