Proton Magnetic Resonance Spectroscopy in Multiple Sclerosis

Synopsis

Proton magnetic resonance spectroscopy (¹H-MRS) provides tissue metabolic information in vivo. This article reviews the role of MRS-determined metabolic alterations in lesions, normal appearing white matter, gray matter, and spinal cord in advancing our knowledge of pathological changes in multiple sclerosis (MS). In addition, the role of MRS in objectively evaluating therapeutic efficacy is reviewed. This potential metabolic information makes MRS a unique tool to follow MS disease evolution, understanding its pathogenesis, evaluating the disease severity, establishing a prognosis, and objectively evaluating the efficacy of therapeutic interventions.

Introduction

Conventional magnetic resonance imaging (cMRI) is exquisitely sensitive in visualizing multiple sclerosis (MS) lesions in brain and spinal cord, but has limited pathologic specificity. Also, cMRI is limited in the detection of subtle, disease-related changes in the normal-appearing white matter (NAWM)¹. This limitation can be overcome to some extent by combining MRI with magnetic resonance spectroscopy (¹H-MRS) which allows detection of tissue biochemical changes for improved pathologic specificity. Since biochemical changes precede anatomical changes, MRS detects tissue pathologic changes even before the appearance of lesions on cMRI². The unique biochemical information provided by ¹H-MRS also complements the information from advanced MRI techniques such as magnetization transfer (MT) and diffusion tensor (DT) MRI in providing a more detailed and specific pathological information. This in turn allows to follow MS disease evolution, better understand its pathogenesis, evaluate the disease severity, establish a prognosis, and objectively evaluate the efficacy of therapeutic interventions^3-5.

This review is divided into three major sections: 1) brief introduction to MRS, 2) review of MRS and its application to MS lesions, NAWM, gray matter, and spinal cord, and 3) role of MRS in clinical trials. Because of the large number of publications on MRS in MS, wherever possible, we have referenced more recent and relevant review articles.

Magnetic Resonance Spectroscopy

In contrast to MRI, which mainly relies on proton (¹H), MRS can be performed using variety of nuclei. Of all these nuclei, ¹H and ³¹P are most commonly employed for MRS studies. For a number of reasons that include sensitivity and hardware considerations, ¹H-MRS is the common choice for investigating neurological disorders. ¹H-MRS provides access to a large number of biomolecules or metabolites such as N-acetylaspartate (NAA; some authors refer to it as NA to acknowledge that the NAA peak has contributions from N-acetylaspartate and other N-acetyl moieties such as N-acetyl aspartyl glutamate [NAAG]. In this review NA and NAA are used interchangeably), choline (Cho), creatine (Cr), myoinositol (mI), glutamate (Glu) and glutamine (Gln), macromolecules, lipids and lactate (Lac). The positions of metabolite peaks in the spectrum, expressed as parts per million (ppm), are independent of the magnetic field strength. NAA is an amino acid derivative synthesized in neurons and transported down axons and its main resonance appears at 2.02 ppm. NAA is generally thought to be a specific marker of neurons, axons, and dendrites⁶. On this basis, reduced NAA concentration is generally interpreted as neuronal/axonal dysfunction and/or loss. This has been confirmed by ¹H-MRS and histology studies on biopsied samples⁷. In general, Glu and Gln are not resolved well even at 3.0 T and combination of resonances from these two is commonly referred to as Glx. The resonances from Glx appear between 2.1 and 2.4 ppm. While some studies have focused on the evaluation of the total Glu and Gln levels under pathological conditions, other studies have employed spectral editing methods to resolve the Glu resonance. Recently Hurd et al.⁸ have used a modified conventional spectral localization technique in which data is collected over multiple echo times (TE). This method detects a single-line Glu resonance at 2.35 ppm. Srinivasan et al.⁹ have shown elevated Glu concentrations in acute lesions (P = 0.02) and NAWM (P = 0.03), with no significant elevation in chronic lesions (P = 0.77) in MS patients compared to normal subjects. The primary resonance of Cr occurs at 3.0 ppm and has contributions both from creatine and phosphocreatine. Elevated Cr levels may represent gliosis⁷. Cho signals at 3.2 ppm have contributions from multiple molecules that include phosphorylcholine, glycerophosphorylcholine, and choline plasmalogen and a very minor contribution from acetylcholine and choline¹⁰. Cho peak appears to reflect cell-membrane metabolism³. Also, elevated Cho concentration represents heightened cell-membrane turnover as seen in demyelination, remyelination, inflammation and gliosis in MS patients³. mI exhibits two resonances, at 3.5 ppm and 4.06 ppm. Due to its proximity to tissue water resonance, the amplitude of the 4.06-ppm peak is affected by the degree of water suppression and is not considered to be a reliable indicator of mI concentration. Brain osmolyte mI appears to be glia specific and it is not found in neurons¹¹. Also, mI is a precursor of phospholipid membrane constituents, and its concentration is affected by the formation and breakdown of myelin. Lac is generally seen as a doublet that resonates at 1.33 ppm. It is the end product of anaerobic glycolysis and is not commonly observed in normal brain. It is increased in acute MS lesions and is related to macrophage activation after membrane breakdown. Lipids along with macromolecules (referred to as lipids for brevity) appear as broad peaks and resonate between 0.8 and 1.5 ppm. In healthy tissues, there should be very weak lipid peaks in the spectrum unless contaminated by subcutaneous fat from non-neural tissues. These peaks have been reported in MS and thought to represent demyelination/remyelination². By measuring the concentration changes in brain metabolites, MRS has provided evidence of early pathology and myelin damage/repair in MS that is not evident on cMRI^{12, 13}.

Acquisition

¹H-MRS can be acquired with spatial information preserved (localized ¹H-MRS), as is most commonly done, or obtain from the whole brain by sacrificing spatial information¹⁴. Point resolved spectroscopy (PRESS)¹⁵ and stimulated echo acquisition mode (STEAM)¹⁶ are the two most commonly used localization techniques in ¹H-MRS. Spectral contamination from extrameningeal tissues is minimized by suppressing the signals from the outer volume¹⁷. Figure 1 demonstrates the placement of eight outer volume suppression bands. The PRESS localization provides better signal-to-noise ratio (SNR) compared to STEAM technique. However, STEAM allows shorterTE- than PRESS.

Figure 1.

Application of outer volume suppression bands to minimize extrameningeal tissue contamination. Left: Eight outer volume suppression bands on one of the five images (each, 3 mm thickness) that represent the spectroscopic volume of interest (15 mm thickness); right: localized image(15 mm slice thickness with the application of the suppression bands) from which ¹H-MRSI is acquired.

Localized ¹H-MRS is acquired in either a single voxel or multi-voxel mode. In single voxel MRS, spectral data are acquired from one location at a time. The volume of this region is typically 1 to 8 cm³. This approach involves shorter acquisition and processing times. High-quality spectrum is relatively easy to acquire in this mode. Multi-voxel mode, also referred to as ¹H-MRS imaging (¹H-MRSI) or chemical shift imaging (CSI) allows simultaneous acquisition of ¹H-MRS data from multiple voxels and allows mapping regional distribution of metabolite concentrations and generate metabolic images. Multi-voxel MRS can be acquired in 2D or 3D and generally offers superior spatial resolution (< 1 cm³) compared to single voxel. Generally, ¹H-MRSI is acquired from a volume that is prelocalized using either PRESS or STEAM. It is also possible to acquire ¹H-MRSI without any prior volume or slab localization (see, for example, Sharma et al.¹⁸). ¹H-MRSI involves relatively long acquisition times, which, however, can be considerably shortened by reduced k-space encoding¹⁹. One way of accelerating ¹H-MRSI acquisition is to employ fast spectral-spatial encoding by using echo-planar-based techniques^20,21. With multi-slice and multi echo sequences, it is possible to acquire spectra from whole brain²¹ within reasonable scan times. Combining EPI-based techniques with recent advances in parallel MRI, such as sensitivity encoding (SENSE)²² or generalized autocalibrating partially parallel acquisitions (GRAPPA)²³ with radiofrequency (RF) coil arrays can further accelerate ¹H-MRSI data acquisition^24-26. However, the data acquired with these approaches generally suffers from reduced SNR. Determination of absolute metabolite concentration is also somewhat problematic with parallel acquisition schemes.

¹H-MRS can be acquired at either long or short TE. Long TE ¹H-MRS provides a well-defined baseline by suppressing peaks arising from short T2 metabolites, such as lipids and macromolecules, and makes spectral quantification simpler and more robust. Long TE ¹H-MRS may be preferable if the interest is in the detection of the three major resonances: NAA, Cr, and Cho. Because of their relatively short T2 relaxation times, signals from metabolites, such as Glx, lipids, macromolecules and other myelin breakdown products can be detected only at short TE. Even though the broad spectral baseline in short TE ¹H-MRS makes the spectral quantification more difficult, this approach has the potential to provide important information about altered tissue biochemistry that may not be available at long TE⁴. There is an increasing interest in short TE ¹H-MRSI for visualizing short T2 metabolites, which should result in improved pathological characterization of tissues³. As an example, single voxel spectra acquired at 3 T with short and long TE, with signal mostly localized to white matter of normal brain, are shown in Figure 2.

Figure 2.

Single voxel spectra with short TE (=35 msec) and long TE (=144 msec) acquired from white matter region of a normal brain at 3.0 Tesla. The large number of metabolite peaks with broad baseline can be observed in the short TE spectrum (top). In contrast, long TE spectrum (bottom) has relatively flat baseline and fewer metabolite peaks. See text for abbreviations

The macromolecular and lipid resonances exhibit significant overlap with the other metabolites. By nulling the metabolite resonances with an inversion recovery sequence, it is possible to improve the detection of these short TE resonances. This method was employed by Mader et al. ²⁷ for separating lipids from other metabolites in acute MS lesions.

While localized techniques can be used to acquire spectrum from the volume of interest (VOI), non-localized sequence can provide MRS from the entire head. The strong broad lipid signals arising from non-brain tissues obscure the resonances from other metabolites. However, by subtracting the data acquired with and without NAA nulling (through an inversion recovery sequence), it is possible to remove/minimize strong lipid signals and visualize resonances from other metabolites¹⁴. Because NAA is restricted to neurons/axons, it is implicitly localized to brain. However, the presence of other resonances in non-brain tissues, compromises the quantification of metabolites such as Cr. Thus this approach is reliable only when quantifying the whole brain NAA (WBNAA) concentration¹⁴. Due to severe magnetic field inhomogeneities present at air-tissue interfaces this technique captures about 90% of the NAA signal intensity in the brain²⁸. Albeit WBNAA measurements do not provide any information about the spatial distribution of metabolite concentrations, this approach eliminates image registration problems from scan-to-scan in serial studies. The reproducibility of WBNAA levels across institutions and MR scanners and field strengths has been shown to be satisfactory²⁹. WBNAA measurements can be used to quantify the global neuronal and axonal injury to monitor the effect of neuroprotective agents in MS ³⁰.

Quantification

The inherently quantitative nature of MRS allows objective monitoring of disease state and response to therapeutic intervention. However, this requires accurate spectral quantification. Expressing the metabolite levels as a ratio relative to Cr is one of the most common and simplest ways of spectral quantification. However, the interpretation of the metabolite ratios becomes equivocal if the concentration of Cr varies with pathology². In addition, representing brain metabolite concentrations as ratios does not provide complete information and could lead to significant errors^{31, 32}. Therefore absolute quantification of metabolite concentrations is highly desirable for proper interpretation of the MRS data. Nevertheless the NAA/Cr ratio is commonly used in MS ³³. Among the methods for estimating the absolute metabolite concentrations, use of internal water as a standard has been shown to provide acceptable accuracy and inter-laboratory reproducibility^{2, 34}. However, this method depends on knowledge of water distribution and relaxation times in different tissues. Absolute concentrations can also be measured using the LCModel.³⁵ This method analyzes the in vivo spectrum as a linear combination of spectra from individual metabolites contained in a solution (model spectrum). The model spectrum is acquired using the same sequence as that used for in vivo spectra and provides the maximum prior knowledge for the spectral analysis. LCModel uses a nearly model-free constrained regularization method that automatically accounts for the fitted baseline and spectral lineshape to be consistent with the data³⁶.

Usually, the ¹H-MRSI quantification procedures involve extensive computational time, and in some cases, significant human intervention. Currently, the most commonly used quantification methods are based on line fitting using a non linear least square optimization, such as the Levenberg-Marquardt algorithm^{35, 37-39}. The relatively long computational time becomes even more problematic when considering 3D ¹H-MRSI and scans from multi-center clinical trials where a large amount of data needs to be processed. For ¹H-MRSI to become a routine clinical tool, complete automation of quantification procedures and reduction of processing times close to real-time are necessary. The processing time could be reduced by using high performance computing environment. However, this is expensive, involves development of complicated algorithms, and is not an option in routine laboratory/clinical settings. Another approach is to develop fast methods, such as those based on artificial neural networks (ANN). ANN has been used to develop automated methods to quantify ¹H-MRS data^{40, 41}. However, these techniques are limited to either single voxel ¹H-MRS or magnitude spectral data acquired at long TE using lineshapes that may not be completely appropriate⁴². Recently, Bhat et al.⁴³ have employed radial basis function neural networks (RBFNN) to demonstrate the feasibility of quantifying short TE, phased ¹H-MRSI. This method is, however, limited to ratios, and thus does not allow absolute concentrations to be measured.

A number of excellent software packages, both free and commercial, are available for spectral quantification. The most commonly used analysis packages include LCModel ³⁵, jMRUI ⁴⁴, and MIDAS ⁴⁵. Most of these packages involve minimal human intervention and are capable of providing absolute concentrations, but involve long processing times. Different analysis methods and their strengths and weaknesses in computing absolute brain metabolite concentrations were recently reviewed ³¹.

Due to relatively large spectroscopic voxels, spectra have contributions from different types of brain tissues, particularly cerebrospinal fluid (CSF). For accurate estimation of metabolite concentrations, it is important to determine the relative volumes of each tissue that contributes to a given spectroscopic voxel. This requires combining the MRSI data with tissue segmentation, based on the high resolution MRI data³⁷.

Application of MRS to MS

Lesions

There is a general consensus that chronic MS lesions exhibit reduced NAA levels, indicating neuronal/axonal loss. This is particularly true in those lesions that also appear hypointense on T1-weighted images (also commonly referred to as “black holes”⁴⁶). In one study, metabolite concentrations from chronic lesions of patients with relapsing-remitting (RR)MS (n=9), secondary progressive (SP)MS (n=10), primary progressive (PP)MS (n=6), and benign MS (BMS) (n=5) were measured and compared with those from white matter of a normal control group (n=9)⁴⁷. A highly significant reduction in NA (the sum of NAA and NAAG) was found in RRMS, PPMS, and SPMS patients compared to controls. However, in patients with BMS there was no significant difference in NA compared to controls. A significant inverse correlation between NA from lesions in MS patients and disability was observed (r=-0.364, 0.05>P>0.02)⁴⁷. Increased Cr and Cho levels were also observed in lesions, suggesting (a) ongoing gliosis and remyelination in isointense lesions on T1-weighted MR images and (b) membrane turnover (de- and remyelination) ⁴⁸.

Acute MS lesions, as detected by gadolinium (Gd) enhancement, demonstrate a range of abnormalities on ¹H-MRS. These abnormalities include reduced NAA, increased Cho, and presence of lipids^{2, 49-52}. Histopathology on biopsied brain tissues demonstrates reduced NAA levels even in the acute phase of the disease⁵³. This could be the result of axonal loss or reflect reversible axonal dysfunction due to functional impairment^{52, 54-56}. Accordingly, serial MRI (with Gd administration) and ¹H-MRSI studies showed transient changes in NAA levels in some acute lesions, which recovered with time². Narayana et al.² have observed that metabolite levels, including NAA, reach their minimum value when lesion volume reaches its maximum. This suggests that the observed decrease in metabolite concentrations, at least partially, can be attributed to resolution of edema within the lesions and is not solely due to compromised tissue metabolism. Cho levels are shown to increase in acute MS lesions^{2, 51, 57}, as well as Lac and mI resonance intensities ^{52, 58}. The presence of lipids in some, but not all lesions was reported in RRMS subjects by Narayana et al.², suggesting the presence of active demyelination/remyelination.

Normal Appearing White Matter

It is now generally agreed, based on MRS^{3, 12}, MT⁵⁹, and DT⁶⁰ studies that the NAWM in patients with MS is abnormal. By exploiting the large spatial coverage allowed by multi-voxel ¹H-MRS, it has been demonstrated that metabolic abnormalities in MS patients are more diffuse and are not restricted to lesion sites alone^{2, 37, 48, 61-64}. ¹H-MRS studies have clearly demonstrated reduced NAA levels in NAWM, suggesting axonal/neuronal loss/dysfunction as the underlying pathologic substrate. The reduced NAA levels on ¹H-MRS have been confirmed by histopathology as secondary to axonal loss^{7, 65}. In serial ¹H-MRS studies, increased Cho and lipid levels were observed from NAWM regions that subsequently went on to develop MRI-visible lesions^{2, 66}. These observations were consistent with MRI studies that showed alterations in MT ratio (MTR) values prior to appearance of macroscopic lesions on MRI⁶⁷. In addition, based on increased mI⁶⁸, Cr and Cho levels, increased cellularity (gliosis, inflammation) was also postulated to occur in NAWM of MS patients⁴⁸.

In a preliminary study, annual MRSI was obtained for two years from 20 patients and 10 healthy controls to characterize the metabolite changes in early RRMS⁶⁹. The concentrations of NAA, Cr, Cho, mI and Glx were estimated in both NAWM and cortical gray matter (cGM). At baseline, the concentration of NAA was 7% lower in NAWM of MS patients compared to the control group and 8.7% lower in cGM. Tissue metabolite profiles did not significantly change in MS subjects nor differed between MS and healthy controls, with the exception of NAA concentrations that tended to recover from baseline. This study suggests that in the early phases of the disease the neuronal/axonal damage may be at least partially reversible. Leary et al.⁷⁰ have performed single voxel ¹H-MRS of NAWM in PPMS (n=24) patients and in 16 age-matched controls. NAA/Cr was significantly lower in NAWM from patients than in the white matter from controls suggesting that axonal loss occurs in NAWM in PPMS patients and may well be a factor associated with disease progression in this disease phenotype. No significant difference was observed in Cr concentration between patients and controls. A multi-center ¹H-MRSI study of 40 PPMS patients showed significantly lower NAA/Cr compared with healthy volunteers, but no significant difference between lesions and NAWM was observed³³. Twenty four of these patients had evident lipid peaks in non-lesion brain tissue locations. Figure 3 shows, as an example, the presence of strong lipid peaks in the spectra from NAWM of a PPMS patient. In another study, metabolite concentrations were obtained with 3D MRS and the VOI centered on the corpus callosum in 11 RRMS patients and nine controls⁷¹. In NAWM, the concentration of NAA was observed to be 9% lower in patients compared to controls. The same study showed an increase in Cr and Cho by 22% and 32%, respectively. These elevated Cho levels differentiated patients from controls with 100% specificity and higher than 90% sensitivity.

Figure 3.

Abnormal peaks in normal-appearing white matter of a patient with primary progressive multiple sclerosis. Top: Left: One of the five MR images (each 3 mm thick) of the cross-section of the brain location from where the ¹H-MRSI data acquired. Right: Collapsed image representing the spectroscopic volume of interest (thickness=15 mm). The small grid on the collapsed image represents the spatial location shown in the bottom panel. Strong lipid peaks can be observed in these spectra. The spectra were acquired at TE of 30 ms.

Suhy et al.⁷² compared metabolite levels in PPMS (n=15) and RRMS (n=13) patients with those of controls (n=20). Cr in NAWM of PPMS patients was higher compared to controls (P=0.035), and RRMS patients (P=0.038). Also, Cr increased in focal MRI lesions from PPMS compared to lesions from RRMS (P=0.044) and compared to control white matter (P=0.041). NAA was similarly reduced in NAWM from PPMS and RRMS compared to controls. The increased Cr in NAWM in PPMS relative to RRMS is consistent with the notion that the progressive disability in PPMS may be associated with gliosis and axonal loss, whereas disability in RRMS may be secondary to an increased burden of acute inflammatory lesions and axonal loss⁷².

Data on NAWM in clinically isolated syndrome (CIS) is limited. In 14 of 42 patients with a single acute episode of optic neuritis, Tourbah et al.⁷³ reported decreased NAA, increased Cho, and the presence of lipids. In 96 patients with CIS suggestive of MS, Fernando et al.⁷⁴ reported increased concentration of mI relative to controls, a finding which is suggestive for the presence of gliosis. No significant difference in other metabolites was observed. In a prospective study on patients with CIS suggestive of MS, Wattjes et al.^{75, 76} studied 31 patients and 20 controls. These authors observed 8.1% decrease in NAA (P= 0.012) in the NAWM of patients relative to controls. However, changes in Cr, Cho, and mI were not observed.

Gray Matter

Cortical pathology in MS is well recognized^{77, 78}. For a number of reasons GM lesions are difficult to visualize on MRI. Using advanced pulse sequences, such as double inversion recovery ^{79, 80} and phase sensitive inversion recovery sequence, it is possible to display some, but not all, cortical lesions ^{81, 82}. Involvement of GM has been demonstrated by MTR⁸³, DTI^{84, 85}, and ¹H-MRS studies^{18, 68, 86, 87}.

¹H-MRS of cGM is technically challenging because of its proximity to CSF and partial volume averaging from white matter⁸⁸. In addition, many of the MRS studies that employ outer volume suppression bands for minimizing contributions from non-neural tissues make it difficult to acquire MRS from the cGM that is close to outer surface of the brain (see Figure 1). In spite of these limitations, MRS studies of cGM have been reported by a few groups. For example, Sharma et al.¹⁸, by combining a double inversion recovery sequence, in which both CSF and white matter were suppressed, demonstrated abnormalities in the cGM close to the midline fissure in MS patients using MRSI. By combining MRSI with image segmentation, this study has minimized contributions from WM and CSF to the VOI and showed in 52 RRMS patients marked lipid peaks arising from the normal appearing GM, consistent with the presence of active demyelination/remyelination. However, no significant changes in major metabolite concentrations were observed compared to controls, suggesting no significant neuronal loss. The lack of significant changes of metabolites in cGM is consistent with another more recent report⁸⁹.

Using short TE MRSI in 16 RRMS patients with short disease duration (mean disease duration of 1.8 years; range 0.6-2.8 years), Kapellar et al.⁸⁶ reported lower NAA (P = 0.01) and mI (P = 0.04) in the cGM relative to controls. The reduced NAA in cGM suggested mild, but widespread, neuronal dysfunction or loss early on in the course of the disease⁸⁶. In another study from the same group of 24 clinically early (median EDSS (Expanded Disability Status Scale) 1.2; range 0.0-3.0) RRMS patients, Chard et al.⁶⁸ reported significantly reduced NAA, Cho, and Glx. They also observed significant correlations between MS functional composite scores and the metabolite concentrations in cGM: Cr (r(s) = 0.524, P = 0.009), cGM Glx (r(s) = 0.580, P = 0.003). However, no correlation was observed between clinical disability and the level of NAA, suggesting that reduced NAA reflects neuronal dysfunction, rather than neuronal loss early on in RRMS.

GM NAA deficits were found in the progressive form of disease, which, however, did not significantly correlate with EDSS⁹⁰. In another study, absolute concentrations of NAA, Cr, and Cho were measured in the occipital-parietal cortex of RRMS (n=15) and SPMS (n=15) patients and healthy subjects (n=8)⁹¹. Significantly lower values of NAA, Cr and NAA/Cr ratio were detected in SPMS patients than in RRMS and control subjects (P<0.01), suggesting that the pathological process in MS extends into cGM, particularly in the progressive form of the disease. Similar observations were reported in a more recent study on a small MS cohort⁸⁷.

The levels of metabolites in the very early stage of the disease appear to be dependent on lesion activity. In CIS suggestive of MS, Au Duong et al.⁸⁸ reported that the metabolite levels in GM are associated with the presence of enhancing lesions: when absent, there was no statistically significant difference in the GM metabolite levels between patients and controls; when present, a relative decrease in NAA level and an increase in Cho level were observed. This suggests that metabolite alterations seen in the GM in the early phase of the disease may be secondary to the inflammatory process, rather than to ‘primary’ neuronal dysfunction.

MS also affects deep GM structures^{92, 93}. ¹H-MRS studies of the thalami were performed in RRMS (n=14) patients and age-matched healthy controls (n=14). Average NAA concentration in the thalami of RRMS patients was approximately 11% lower relative to controls (p<0.05). In addition, about 25% lower mean normalized thalamic volumes was observed in patients compared to controls (p<0.005). Decreases in thalamic NAA concentrations strongly correlated with thalamic volume loss⁹⁴. The reduction of both NAA concentration and thalamic volume suggests a neurodegenerative component to MS that is increasingly recognized in the disease ⁹⁵.

Whole brain NAA

Recently, WBNAA as a marker of overall axonal/neuronal loss in the brain has been proposed⁹⁶. The concentration levels of WBNAA and normalized brain volumes were significantly lower in PPMS patients than in healthy controls⁹⁷. Also, WBNAA values and normalized brain volumes were not related suggesting that MRS and atrophy assessment may provide complementary information about the extent of brain damage in PPMS. Axonal/neuronal damage in PPMS patients seems to occur, at least partially, independent of the burden of MRI-visible lesions⁹⁷. In another study, WBNAA levels were compared with atrophy as a function of disease duration in a cohort of 42 RRMS patients. WBNAA levels were found to decrease 3.6 times faster than atrophy, suggesting that neuronal/axonal dysfunction precedes atrophy⁹⁸, and that WBNAA may be a more sensitive indicator of disease progression than lesion load or atrophy.

Although axonal pathology has been known to occur in MS, it is less clear how early in the course of disease axonal damage starts to appear, as well as its relation to MRI-visible lesion load. To assess this early axonal pathology, Filippi et al.⁹⁹ estimated the WBNAA levels in 31 patients with CIS suggestive of MS. The mean WBNAA concentration was significantly lower in patients compared with the controls. It was not significantly different between patients with and without enhancing lesions at baseline MRI or between patients with and without lesion dissemination in time. WBNAA concentrations and lesion volumes were not correlated. These findings, consistent with both histology and localized ¹H-MRS studies, suggest the presence of widespread axonal pathology in the earliest clinically symptomatic stage of MS.

Spinal Cord

Involvement of spinal cord in MS and its potential role in patient disability has been recognized for some time^{100, 101}. Post-mortem examination of MS patients reported substantial atrophy and axonal loss in the lateral columns at C3 and T2¹⁰². However, cord atrophy and total axonal number are not strongly correlated¹⁰³, suggesting that cord atrophy may underestimate axonal loss and may not provide a valid marker of disease progression¹⁰⁴. ¹H-MRS may provide valuable information about true axonal damage in spinal cord in MS patients. In vivo ¹H-MRS of spinal cord is quite challenging due to magnetic field inhomogeneities around the cord, physiological movements, and small cross sectional size. Therefore, it is not surprising that relatively few ¹H-MRS studies of the spinal cord have been performed. However, Marliani et al.¹⁰⁵ demonstrated the feasibility of performing quantitative MRS in the cervical region on a clinical 3 T system.

¹H-MRS of normal appearing spinal cord in MS patients showed significantly reduced NAA and altered concentration levels of other metabolites compared to healthy controls¹⁰⁶. In one study, MRS was used to investigate the degree of neuronal damage in the cervical cord in MS patients. Spectra were acquired from spinal cord and brain in 11 patients and 11 controls. A 32% reduction of NAA concentration was observed in patients relative to controls (P < 0.05), indicating significant neuronal/axonal injury. Additionally, significant cord atrophy was observed in MS patients (15%, P < 0.001). No correlations between clinical measures and cord atrophy or brain lesion volume were found; however, spinal cord NAA correlated with the cerebellar subscore of the EDSS (P < 0.005). ¹H-MRS demonstrated cellular damage within the cord over and above the tissue atrophy seen on MRI¹⁰⁴.

In another study, Ciccarelli et al.¹⁰⁷ acquired single-voxel ¹H-MRS from the cervical cord of 14 patients with at least one lesion between C1 and C3. Thirteen age- and sex-matched control subjects were included in the study. Patients showed reduced NAA relative to controls. In patients, significant correlations between (i) EDSS and mI, Cho, Cr, and (ii) 9-hole peg test (9-HPT) and Cr were observed. The concentration of mI was independently associated with the EDSS, while Cr, NAA were independently associated with the 9-HPT. While the limited ¹H-MRS studies on cervical cord have provided useful information about tissue damage, the full potential of ¹H-MRS in spinal cord in MS patients has yet to be exploited.

Evaluation of Treatment Efficacy

Immunomodulatory therapies, including glatiramer acetate (GA), interferon (IFN)-β, and natalizumab, are shown to be effective in reducing MRI-measured disease activity^108-111. Because of its inherent quantitative nature, MRS is ideally suited for evaluating the efficacy of treatment. The effect of immunomodulatory treatments on brain metabolites for evaluating treatment efficacy was studied in multiple, but small patient groups. In an open-label, non-randomized pilot study of GA treatment in 15 RRMS patients, Narayanan et al.¹¹² measured NAA/Cr in a large VOI centered in the corpus callosum prior to treatment and one year after. The untreated and treated groups had similar mean baseline NAA/Cr and EDSS. At one year, the GA-treated group showed no change in the NAA/Cr ratio. However, the untreated group had a small (3.5%) but significant (p=0.015) reduction in NAA/Cr. During this short period of treatment, GA was shown to stabilize the levels of brain NAA which is consistent with the putative neuroprotective effect of the drug, GA.

In another pilot study, Khan et al.¹⁰⁹ obtained ¹H-MRS from 18 RRMS patients before and after treatment with GA. These patients were followed annually for two years. A small group of four treatment naïve RRMS patients was also studied. ¹H-MRSI was acquired from a large VOI that contained both corpus callosum and adjacent white matter. The VOI included both lesions and NAWM. The mean NAA/Cr was measured from the whole VOI and NAWM. After two years, NAA/Cr in the GA treated group increased significantly (10.7%) in the VOI, and by 7.1% in the NAWM. In the untreated group, NAA/Cr decreased by 8.9% at two years in the VOI and 8.2% in the NAWM. This study showed that treatment with GA may lead to axonal metabolic recovery and protection from sub-lethal axonal injury in RRMS patients. Long term results of this ongoing study after 4 years of annual brain MRS examinations were reported recently¹¹³. Compared to baseline, at year 4, patients receiving continuous GA therapy showed an overall increase of 12.7% in NAA/Cr (P = 0.03) within the entire VOI and by 9.6% (P = 0.04) in the NAWM. Three patients in the control group who began therapy with GA during the course of the study showed similar increases in NAA/Cr after the first year of therapy. These data support the notion of a possible sustained effect of GA in RRMS and the feasibility of employing MRS in long-term investigative studies of MS¹¹³.

In a multi-center longitudinal MRSI study, the efficacy of GA treatment in 58 PPMS patients was investigated annually for three years¹¹⁴. This patient population was a subcohort of the PROMiSe trial patients¹¹⁵. Quantitative NAA/Cr and Cho/Cr were compared between GA-treated and placebo-treated PPMS patients. This study failed to demonstrate a significant effect of GA on the metabolite concentrations. There were no significant changes in metabolite concentrations in untreated PPMS patients over three years. These findings are different from those in RRMS described above and may suggest that substantive decreases in NAA/Cr may have occurred at an earlier stage of disease in PPMS and treatment with GA may not result in improvement of the metabolite profile¹¹⁴.

Longitudinal MRS studies have been carried out in 10 RRMS patients who, after baseline examination, received interferon β-1b. Spectra were acquired up to 34 months at different time points (from 8-20 time points), and absolute concentrations of NAA, Cr, and Cho were determined in a large non-enhancing lesion and contralateral NAWM¹¹⁶. The concentrations of Cho and Cr were found to be higher in MS than in healthy controls ¹¹⁶. These studies, except in one out of 10 patients, failed to demonstrate any effect of interferon β-1b on the metabolic levels.

¹H-MRS has also been used to investigate the modifications of brain metabolites, in the initial phase of interferon β-1a treatment¹¹⁷. This study was performed on 5 RRMS patients who were treated with intramuscular IFN β-1a for six months and 5 untreated patients. Patients were evaluated at the beginning of the study and in the first, third, and sixth month of treatment. In white matter lesions, NAA, Cho, Cr, and mI peaks did not vary significantly over the entire period of study in the untreated group. In the treated group, there was a significant increase in Cho at the first month relative to the pretreatment period, and continued to increase at months 3 and 6 (p<0.001). A slight but not statistically significant increase in Cho was also found in the NAWM from the patients treated with IFN β-1a.

In a small pilot study, ¹H-MRS was performed in 10 RRMS patients before and after one year of treatment with subcutaneous interferon β-1b. NAA/Cr was measured in a large central brain volume¹¹⁸. These measurements were compared with those from six untreated patients selected for similar range of EDSS scores and mean NAA/Cr at baseline. NAA/Cr in the treated group showed an increase of 5.5% at 12 months of therapy, while this ratio decreased in the untreated group, but not significantly. NAA/Cr was significantly higher in the treated group at 12 months than in the untreated group¹¹⁸, suggesting an effect of interferon β-1b in restoring neural integrity.

Unfortunately, many of these studies are based on small patient populations followed over relatively short intervals. Lack of standardized acquisition and analysis protocols makes it very difficult to evaluate the results critically.

Conclusions

¹H-MRS is a valuable tool that could contribute in objectively following the evolution of MS, to the understanding of its pathogenesis, evaluating disease severity, establishing prognosis, and assessing the efficacy of therapeutic interventions. Some of the most compelling evidences that axonal loss is a major cause of disability in MS has come from ¹H-MRS studies which show reduced NAA levels in brain of these patients. ¹H-MRS studies have shown that axonal damage is an early event that occurs before the formation of MRI-visible lesions. However, a number of technical factors that include poor SNR, long acquisition times, poor spatial resolution, limited spatial coverage, and complex data processing have so far limited the use of ¹H-MRS in routine clinical practice. Recent developments of high field MRI scanners for improved SNR and spectral resolution, introduction of parallel imaging, fast analysis techniques, and the availability of free analysis tools should greatly facilitate a more widespread use of ¹H-MRS in the diagnosis and management of MS. Another aspect of MRS that needs to be addressed is the standardization of both acquisition and analysis protocols. A first step towards achieving the standardization, based on single voxel MRS, has recently been proposed¹³. While this is an appropriate first step, standardized protocols that include multi-voxel MRS for increased spatial coverage and exploit the full potential of MR hardware and software are needed.