Axonal injury in early multiple sclerosis is irreversible and independent of the short-term disease evolution
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Abstract
Objective: To define the nature and the temporal evolution of neuronal/axonal injury in patients at the earliest clinical stage of multiple sclerosis (MS), using whole brain N-acetylaspartate (WBNAA) proton MR spectroscopy (1H-MRS).
Methods: Thirty-five patients at presentation with clinically isolated syndromes (CIS) and MRI evidence of disease dissemination in space were studied. The following scans of the brain were acquired within 3 months from the onset of the disease and after 12 months: 1) dual-echo; 2) WBNAA 1H-MRS; 3) pre- and postcontrast T1-weighted. The same scans were obtained in 12 age-matched healthy subjects, without contrast administration. In patients, conventional MRI scans were also repeated 3 months after the first scanning session, to assess the presence of early disease dissemination in time (DIT).
Results: Over the study period, 24 patients showed MRI evidence of disease DIT, thus fulfilling the criteria for a diagnosis of MS. The average WBNAA amount was lower in CIS patients than in controls both at baseline (13.7 vs 16.9 mM, p < 0.001) and at 1-year follow-up (12.6 vs 16.2 mM, p < 0.001), but the average yearly percentage change of WBNAA did not differ between the two groups. No MRI or 1H-MRS quantities were significantly associated with the disease DIT over the study period.
Conclusion: Irreversible brain damage associated with axonal dysfunction occurs at a very early stage in patients with clinically isolated syndromes, but it does not seem to be related with the disease evolution in the subsequent short-term period.
Following convincing evidence of a beneficial role of early immunomodulating treatment1,2 in patients with initial findings suggestive of multiple sclerosis (MS), the identification of patients at presentation with clinically isolated syndromes (CIS) with a high risk of subsequent conversion to MS has become an issue of paramount relevance.3 The prognostic value of conventional MRI findings in CIS is well known, but it remains suboptimal and controversial.4–6 Therefore, several studies using quantitative MR-based techniques have tried to quantify the extent of brain and cord damage beyond T2-visible lesions in CIS, with the ultimate goal to define reliable paraclinical predictors of CIS evolution. However, the results have been conflicting and not exhaustive.7–15
Among quantitative MR-based studies of CIS, those using proton MR spectroscopy (1H-MRS)7,9,10,13 have the potential to provide us with a sensitive and specific marker of neuronal and axonal viability by measuring the amount of N-acetylaspartate (NAA), a metabolite which is localized almost exclusively within neuronal cell bodies and their projections.16 Because of the functional relevance of axonal pathology in MS,17 the estimation of the brain NAA concentration in CIS patients might be useful to better define their prognosis. Using a nonlocalized 1H-MRS acquisition scheme18,19 to quantify the amount of NAA in the whole of the brain (WBNAA), we found that the average WBNAA concentration was significantly reduced in CIS patients when compared with age-matched controls.10 Therefore, we hypothesized that widespread axonal pathology occurs in patients even at the earliest clinical stage of MS. In a previous study,9 a significant increase of myoinositol levels in CIS patients compared to healthy subjects was found, suggesting the presence of reactive gliosis already at this stage of the disease. However, no significant changes of NAA levels in CIS patients were reported by this and other studies.7,9,13
Against this background, the present study was performed with the following aims: to expand our previous findings10 in a larger cohort of CIS patients, to investigate whether WBNAA 1H-MRS-detectable axonal damage is transient or persistent, and to assess whether the WBNAA profile of CIS patients may have a prognostic value for the short-term occurrence of clinical or MRI disease dissemination in time (DIT) leading to a diagnosis of MS.20
Methods.
Subjects.
To be included, patients had to have a CIS within the 3 months preceding study initiation and paraclinical evidence of disease dissemination in space (DIS).20 Appropriate investigations were carried out as necessary to exclude alternative diagnoses. Steroid treatment, if any, had to be concluded at least 3 weeks before the first scanning session. Clinical evaluation comprised neurologic visits with expanded disability status scale (EDSS)21 rating, within 3 days from the acquisition of baseline and follow-up MRI scans. All patients were evaluated by a single neurologist, unaware of the MRI results. In case of symptoms suggestive of a clinical relapse during the study period, patients were instructed to contact the same neurologist for additional visits. Twelve sex- and age-matched healthy volunteers (four men and eight women; mean age: 28.9 years; range: 24-34 years), with a normal neurologic examination, served as controls. All subjects signed a written informed consent prior to study entry and the study design was approved by the local ethics committee.
Image acquisition.
MRI and 1H-MRS were performed using a 1.5-T scanner (Siemens Vision, Siemens, Erlangen, Germany). The following sequences were collected from all subjects during a single MR session: dual-echo turbo spin-echo (SE) (repetition time [TR] = 3,300, echo time [TE] = 16/98, echo train length = 5, 24 contiguous, 5 mm-thick, axial slices with a 256 × 256 matrix and a 250 × 250 mm field of view); 1H-MRS based on a four-step cycle of nonselective 180-degree inversion pulses to obtain WBNAA measurement, following the acquisition scheme described previously;18 and T1-weighted conventional SE (TR = 768, TE = 14, 24 contiguous, 5 mm-thick, axial slices with a 256 × 256 matrix and a 250 × 250 mm field of view) before and after the administration of 0.1 mmol/kg of gadolinium (Gd) (contrast medium was not administered to normal controls). In patients, dual-echo, pre- and postcontrast T1-weighted scans were also obtained 3 months after the first scanning session, to assess the presence of MRI signs of disease DIT.20 In all subjects, the same scan procedure as baseline was repeated after at least 12 months (the mean interval between baseline and 1-year follow-up scans was 13.3 months, range: 11 to 22 months). For both follow-up scans, subjects were repositioned using ad hoc guidelines for MS studies.22
MR analysis.
MRI hardcopies were reviewed in a random order by two observers, unaware of subjects' identity. Lesions were identified and marked by consensus on the proton-density and postcontrast T1-weighted scans. T2-weighted images were always used to increase the confidence in lesion identification. The number and location of T2-hyperintense and Gd-enhancing lesions were evaluated. The scans from individual patients were then ordered chronologically and the fulfillment of MRI criteria for DIS and DIT was assessed.20 Digital MR images were transferred to a workstation (SUN Sparcstation; Sun Microsystem, Mountain View, CA) for T2-hyperintense lesion volume (LV) measurements. These were performed by a single observer, unaware of subject's identity, using a semiautomated segmentation technique based on local thresholds, keeping the marked hardcopies as a reference.23
Using T1-weighted images, both longitudinal (two timepoints) percentage brain volume change (PBVC) and cross-sectional (single timepoint) normalized brain volume (NBV) were estimated. PBVC was estimated using Structural Image Evaluation of Normalized Atrophy (SIENA) and NBV was estimated using the cross-sectional version of SIENA (SIENAX).24 In both methods, the first stage is the extraction of the brain from each input MR image(s), using Brain Extraction Tool. The original images are then registered to a canonical image in a standardized space. In the longitudinal method, to estimate changes between the images, SIENA finds all brain surface edge points using tissue-type segmentation, and then correlates differentiated 1D perpendicular profiles taken around the position of these points in both images. Brain atrophy is quantified by taking the mean perpendicular edge motion over all edge points and converting this into PBVC. The normalizing factor in the conversion is found by a self-calibration step, which involves finding estimated atrophy on an artificially scaled version of one image with respect to itself. In SIENAX, a similar registration process is applied, but instead of a second time point image, standard space average brain and skull images are used. The estimate of brain tissue volume for a subject is then multiplied by the normalization factor to yield the NBV. From each subject, the NBV at baseline and the PBVC between baseline and month-12 scans were obtained.
The 1H-MRS data from each subject and from the reference phantom were transferred to the workstation and processed offline with our custom software (IDL; Research System, Boudler, CO). The NAA peak area was integrated by two operators, by consensual agreement and being unaware of subjects' status as normal controls or patients. It was converted into absolute amount (in mmoles) by scaling against the area of the signal from the reference phantom which contained 15 mmoles NAA (5 mM) in water.18 To correct for the considerable natural interindividual brain size variations, the absolute NAA amount from each individual was divided by the absolute brain volume. This yielded an absolute WBNAA concentration, in mM, which can be compared cross-sectionally and longitudinally.18,19
Statistical analysis.
Group comparisons were analyzed using the Student t test. Univariate correlations were assessed using the Spearman Rank Correlation Coefficient. A univariate logistic regression model was used to test whether there were MRI or 1H-MRS variables predicting the probability to show MRI or clinical evidence of disease DIT at follow-up. To investigate the potential role of WBNAA concentration and of its short-term changes in the workup of individual cases, we classified patients' values for each of these two quantities as abnormal when they were two standard deviations or more below the corresponding mean values obtained from the cohort of healthy subjects.
Results.
Thirty-five patients (7 men and 28 women; mean age: 27.1 years, range: 23-43 years) were studied. Clinical presentations of CIS were optic neuritis in 13 (37%), hemispheric brain syndrome in eight (23%), brainstem syndrome in 10 (29%), and spinal cord syndrome in four (11%) patients. Oligoclonal bands were found in the CSF of 31 patients. One Gd-enhancing lesion was found on the baseline scans of eight patients. By definition, all patients had paraclinical evidence of disease DIS: in 30 patients this was demonstrated by MRI alone and in the remaining five by the combination of MRI findings and the presence of oligoclonal bands in the CSF.20 At follow-up, 24 patients (69%) showed MRI evidence of DIT (10 at month 3 and 14 at month 12), thus fulfilling the criteria for a diagnosis of MS.20 MRI DIT was associated with evidence of clinical DIT over the study period in seven of these patients.
The table reports MRI and 1H-MRS findings in CIS patients and healthy controls. No abnormalities were seen on any of the conventional MRI scans from the controls. At 1-year follow-up, CIS patients showed an (p = 0.024) increase of brain T2 LV. Neither NBV nor PBVC were different between patients and controls (p = 0.86 and 0.32); however, the value of PBVC differed from zero for CIS patients (p = 0.001) but not for controls (p = 0.89). The average WBNAA concentration was lower in CIS patients than in healthy controls, both at baseline (p < 0.001) and at 1-year follow-up (p < 0.001). The mean percentage decrease of WBNAA was 7.3% (SD 13.7) in CIS patients and 4.7% (SD 3.8) in controls (p = 0.52 for the between-group comparison).
Table MRI and WBNAA1H-MRS findings in 35 CIS patients and 12 healthy controls
No significant correlations were found between baseline WBNAA and T2 LV (r = –0.15), NBV (r = –0.11), T2 LV percentage changes at 1-year follow-up (r = 0.11) or PBVC (r = –0.11). WBNAA percentage decrease at 1-year follow-up was not significantly correlated with baseline T2 LV (r = –0.24), WBNAA (r = –0.01), or NBV (r = –0.25). No significant correlations were found between the on-study percentage changes of WBNAA and those of T2 LV (r = 0.29) or PBVC (r = –0.07).
No significant differences for any of the MRI and 1H-MRS-derived quantities at baseline and follow-up or for the corresponding percentage changes during the study period were found between patients with DIS shown by MRI alone and those in whom a combination of MRI and CSF findings was needed to demonstrate DIS, nor between patients with and those without Gd-enhancing lesions on baseline MRI scans (data not shown). There were no MRI or 1H-MRS-derived variables significantly predicting the occurrence of MRI or clinical disease DIT during the follow-up period.
Baseline WBNAA was abnormal in 19 patients (54%) and WBNAA percentage change at 1-year in another 11 patients (31%), whereas both the quantities were abnormal in six patients (17%). CIS patients with abnormal baseline values or on-study percentage changes of WBNAA did not show a higher frequency of MRI or clinical disease DIT during the follow-up period (data not shown).
Discussion.
Post mortem25–27 and in vivo quantitative MR-based studies28 have consistently shown the presence of irreversible damage to axons and neurons in patients with MS, since the early clinical stages of the disease. Using WBNAA 1H-MRS, we confirmed the results of a previous report10 and found, in the brain of CIS patients with MRI features associated with a high risk of developing established MS, a significant neuroaxonal damage has occurred. The novel findings of this study are that such a damage is still present 1 year after the CIS onset and its severity is independent of the disease evolution in terms of clinical or MRI evidence of DIT.20
The actual patterns and severity of brain damage occurring outside T2-visible lesions in CIS patients remain a controversial issue, as demonstrated by the discrepant results obtained by studies using magnetization transfer MRI,8,12–15 diffusion tensor MRI,11,29 and 1H-MRS.7,9,10,13 However, there is an increasing body of evidence suggesting that the extent of normal-appearing brain tissue damage in CIS does not predict the short-term disease evolution. The results of the present study support the latter hypothesis, but are in apparent contrast with those of three other 1H-MRS studies that did not report any significant NAA decrease in the periventricular normal-appearing white matter7,9 and in the corpus callosum13 of CIS patients. In all these studies,7,9,13 however, the use of single-voxel 1H-MRS may have reduced the sensitivity of the technique, which was restricted to small volumes of interest (VOI), typically representing less than 5% of the whole brain parenchymal volume. In addition, estimates of the extent of axonal pathology from single-voxel measurements rely on the assumption that NAA changes in the VOI accurately reflect the status of the entire MS brain.30 In contrast with this, recent evidence31–33 from quantitative MR-based studies supports the notion that the progressive accumulation of gray matter damage is predominant in the early stages of MS and, at least partially, independent of the concomitant pathologic features of the white matter. Against this background, WBNAA 1H-MRS, thanks to its complete brain coverage, might well be able to disclose the presence of a subtle, but widespread and irreversible, neuronal/axonal damage in the brain of CIS patients. Clearly this goes at the price of lacking spatial resolution and of not providing us with information about gray and white matter pathologic features in isolation.
We found that, 1 year after CIS onset, WBNAA was still lower in CIS patients than in age-matched healthy controls. However, a nonsignificant decrease of WBNAA concentration at follow-up was observed, which did not show significant between-group differences too and, therefore, did not change the magnitude of the average reduction observed in patients when compared with controls. These findings indicate that the decrease of WBNAA, which is detectable soon after the CIS onset,10 is persistent and does not merely depend upon sublethal axonal injury34 or upon reversible reduced concentrations in axons attributable to acute inflammatory changes leading to edema.35 Such a decrease most likely reflects the presence of irreversible neuronal and axonal injuries, which are well known to occur since the earliest phases of MS.36 The lack of a significant relationship between T2 LV and WBNAA amount also supports the notion that such an irreversible axonal/neuronal damage is not merely due to the pathologic features of established MRI-visible lesions. The observed further decrease of WBNAA concentrations both in patients and in controls after 1 year, albeit being slightly and not significantly greater in the former than in the latter group, might be due to a technical drift of measurements rather than to an actual progression of the severity of axonal damage in CIS patients. However, during the follow-up period, our scanner underwent regular maintenance without upgrades, reference phantoms for WBNAA quantification correction were prepared de novo and scanned on a monthly basis to avoid any deterioration and the results did not change when corrected for the dates of subjects' scan acquisition (data not shown). These findings indicate that, for a correct interpretation of longitudinal WBNAA measurements, age-matched controls need also to be rescanned in parallel with patients. However, the stability of WBNAA measurements over time warrants further investigation.
There are several reasons why, in the present study, the observed decrease of WBNAA in patients (albeit irreversible) was unrelated to either the occurrence of clinical or MRI-detectable disease activity within 1 year after CIS onset. First, a longer follow-up might lead to different results because the proportion of CIS patients developing MS continues to increase after 10 years4 and the prognostic value of MRI characteristics for the clinical evolution of MS seems to become stronger after longer periods of observation.37 Second, the role played by regional damage in driving the prognosis of CIS38 can not be ascertained by WBNAA measurements. Third, neurodegenerative and inflammatory pathologic features might be dissociated in early MS, with a relative prevalence of the latter aspects, which seem to widely account for the disease evolution after its clinical onset. This hypothesis is supported by the observations that neither magnetization transfer8,12,14,15 nor diffusion tensor11,29 MRI-derived metrics seem to have a stronger prognostic value than T2-visible lesion burden in patients with CIS. Discrepant results have, however, been obtained using other MR measures of irreversible tissue damage, such as brain31,39,40 and cord41 atrophy. The rate of brain atrophy was always found to be significantly greater in patients developing MS than in those without evidence of disease DIT. In all the latter studies, however, part of CIS subjects had normal brain MRI at baseline and, therefore, a low risk of developing MS, who might have accounted for the observed differences. In addition, the rate of atrophy also reflects other degenerative components of MS-related tissue damage, such as demyelination, which, on turn, may not cause changes of WBNAA concentration.
We are aware that the interpretation of these findings has to be cautious because of the limited sample size and the restricted patients' selection criteria. The lack of other longitudinal WBNAA 1H-MRS studies in patients with established MS also makes it difficult to interpret the actual meaning of the observed (absence of) changes of WBNAA profiles in CIS after 1 year of observation. However, a longer follow-up study of this cohort has already been planned to fully ascertain whether WBNAA measurement may help us to identify CIS patients with a poorer prognosis.
Footnotes
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Disclosure: The authors report no conflicts of interest.
Received June 2, 2005. Accepted in final form August 9, 2005.
References
- 1.↵
- 2.
- 3.↵
- 4.↵
- 5.
- 6.
Mikaeloff Y, Adamsbaum C, Husson B, et al. MRI prognostic factors for relapse after acute CNS inflamatory demyelination in childhood. Brain 2004;127:1942–1947.
- 7.↵
- 8.↵
Brex PA, Leary SM, Plant GT, Thompson AJ, Miller DH. Magnetization transfer imaging in patients with clinically isolated syndromes suggestive of multiple sclerosis. AJNR Am J Neuroradiol 2001;22:947–951.
- 9.↵
Fernando KTM, McLean MA, Chard DT, et al. Elevated white matter myo-inositol in clinically isolated syndromes suggestive of multiple sclerosis. Brain 2004;127:1361–1369.
- 10.↵
Filippi M, Bozzali M, Rovaris M, et al. Evidence for widespread axonal damage at the earliest clinical stage of multiple sclerosis. Brain 2003;126:433–437.
- 11.↵
- 12.
Iannucci G, Tortorella C, Rovaris M, Sormani MP, Comi G, Filippi M. Prognostic value of MR and magnetization transfer imaging findings in patients with clinically isolated syndromes suggestive of multiple sclerosis at presentation. AJNR Am J Neuroradiol 2000;21:1034–1038.
- 13.↵
Ranjeva JP, Pelletier J, Confort-Gouny S, et al. MRI/MRS of corpus callosum in patients with clinically isolated syndrome suggestive of multiple sclerosis. Mult Scler 2003;9:554–565.
- 14.
Rovaris M, Gallo A, Riva R, et al. An MT MRI study of the cervical cord in clinically isolated syndromes suggestive of MS. Neurology 2004;63:584–585.
- 15.
Traboulsee A, Dehmeshki J, Brex PA, et al. Normal-appearing brain tissue MTR histograms in clinically isolated syndromes suggestive of MS. Neurology 2002;59:126–128.
- 16.↵
- 17.↵
- 18.↵
- 19.
Gonen O, Catalaa I, Babb JS, et al. Total brain N-acetylaspartate. A new measure of disease load in MS. Neurology 2000;54:15–19.
- 20.↵
- 21.↵
Kurtzke JF. Rating neurological impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983;33:1444–1452.
- 22.↵
Miller DH, Barkhof F, Berry I, Kappos L, Scotti G, Thompson AJ. Magnetic resonance imaging in monitoring the treatment of multiple sclerosis: concerted action guidelines. J Neurol Neurosurg Psychiatry 1991;54:683–688.
- 23.↵
- 24.↵
- 25.↵
Evangelou N, Konz D, Esiri MM, Smith S, Palace J, Matthews PM. Regional axonal loss in the corpus callosum correlates with cerebral white matter lesion volume and distribution in multiple sclerosis. Brain 2000;123:1845–1849.
- 26.
Ferguson B, Matyszac MK, Esiri MM, Perry VH. Axonal damage in acute multiple sclerosis lesions. Brain 1997;120:393–399.
- 27.
- 28.↵
- 29.
Caramia F, Pantano P, Di Legge, S, et al. A longitudinal study of MR diffusion changes in normal appearing white matter of patients with early multiple sclerosis. Magn Reson Imaging 2002;20:383–388.
- 30.↵
Pelletier D, Nelson SJ, Grenier D, Lu Y, Genain C, Goodkin DE. 3-D echo planar 1-HMRS imaging in MS: metabolite comparison from supratentorial vs. central brain. Magn Reson Imaging 2002;20:599–606.
- 31.↵
Dalton CM, Chard DT, Davies GR, et al. Early development of multiple sclerosis is associated with progressive grey matter atrophy in patients presenting with clinically isolated syndromes. Brain 2004;127:1101–1107.
- 32.
Tiberio M, Chard DT, Altmann DR, et al. Gray and white matter volume changes in early RRMS. A 2-year longitudinal study. Neurology 2005;64:1001–1007.
- 33.
- 34.↵
De Stefano N, Narayanan S, Matthews PM, Francis GS, Antel JP, Arnold DL. In vivo evidence for axonal dysfunction remote from focal cerebral demyelination of the type seen in multiple sclerosis. Brain 1999;122:1933–1939.
- 35.↵
- 36.↵
Kuhlmann T, Lingfeld G, Bitsch A, Schuchardt J, Bruck W. Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain 2002;125:2202–2212.
- 37.↵
Rovaris M, Agosta F, Sormani MP, et al. Conventional and magnetization transfer MRI predictors of clinical multiple sclerosis evolution: a medium-term follow-up study. Brain 2003;126:2323–2332.
- 38.↵
- 39.
Brex PA, Jenkins R, Fox NC, et al. Detection of ventricular enlargement in patients at the earliest clinical stage of MS. Neurology 2000;54:1689–1691.
- 40.
Dalton CM, Brex P, Jenkins R, et al. Progressive ventricular enlargement in patients with clinically isolated syndromes is associated with the early development of multiple sclerosis. J Neurol Neurosurg Psychiatry 2002;73:141–147.
- 41.↵
Brex PA, Leary SM, O'Riordan, JI, et al. Measurement of spinal cord area in clinically isolated syndromes suggestive of multiple sclerosis. J Neurol Neurosurg Psychiatry 2001;70:544–547.
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