†These authors contributed equally as joint first-authors.
Academic Editor: Luis Puelles
Magnetic resonance spectroscopy (MRS) has contributed important academic insights in motor neuron diseases (MNDs), particularly in amyotrophic lateral sclerosis (ALS). Over the past three decades momentous methodological advances took place, including the emergence of high-field magnetic resonance imaging (MRI) platforms, multi-voxel techniques, whole-brain protocols, novel head-coil designs, and a multitude of open-source imaging suites. Technological advances in MRS are complemented by important conceptual developments in MND, such as the recognition of the importance of extra-motor brain regions, multi-timepoint longitudinal study designs, assessment of asymptomatic mutation carriers, description of genotype-associated signatures, and the gradual characterisation of non-ALS MND phenotypes. We have conducted a systematic review of published MRS studies in MND to identify important emerging research trends, key lessons from pioneering studies, and stereotyped shortcomings. We also sought to highlight notable gaps in the current literature so that research priorities for future studies can be outlined. While MRS remains relatively underutilised in MND compared to other structural, diffusivity and functional imaging modalities, our review suggests that MRS can not only advance our academic understanding of MND biology, but has a multitude of practical benefits for clinical and pharmaceutical trial applications.
Motor Neuron Diseases (MNDs) are a heterogeneous group of neurodegenerative conditions with divergent disability profiles , survival characteristics  and pathological patterns [3, 4]. Imaging studies in MNDs are dominated by structural [5, 6], diffusion  and fMRI studies [8, 9], despite landmark magnetic resonance spectroscopy (MRS) studies, confirming medication effects , detecting presymptomatic changes , describing alterations in cortical  and subcortical  regions, in cross-sectional and longitudinal studies [14, 15].
Complementary to structural gray matter (GM) and white matter (WM) magnetic
resonance imaging (MRI), MRS allows non-invasive and in vivo
quantification of different molecular constituents’ (metabolites) concentration
in brain regions based on their characteristic frequencies resonance in strong
magnetic fields . Even though earlier studies focused on the 31P nucleus for
the measurement of energy metabolites (e.g., phosphocreatine and ATP) , the
vast majority of brain MRS in vivo studies use proton MRS (1H-MRS) which
became prevalent due its higher sensitivity and convenience in being performed on
most clinical MRI machines . 1H-MRS detects radiofrequency signals arising
from hydrogen nuclear spins within tissue metabolites; these signals have
chemically specific frequencies which are determined by the chemical environment
of the hydrogen spins. The MRS signals are separated in the MR spectrum along
chemical lines, i.e., chemical shift dimension. The spectrum is a plot of signal
intensity (proportional to metabolite concentration) against the frequencies of
resonance. The latter is reported in field-independent units, i.e., parts per
million of the proton frequency (ppm). The most common metabolites that can be
observed using brain 1H-MRS are lactate (Lac), lipids, alanine, N-acetyl-asparate
(NAA), creatine (Cr), choline (Cho), myo-inositol (mIns), glutamate (Glu),
glutamine (Gln), and
MND is a clinically heterogenous, inexorably progressive neurodegenerative condition. The term MND is an umbrella term which encompasses a multitude of phenotypes with unique clinical features, distinctive disability profiles, divergent progression rates and different survival profiles [3, 4, 23]. Pharmaceutical trials in MND have overwhelming targeted ALS cohorts to data and often rely on clinical scales, respiratory function and survival as study end-points instead of quantitative biomarkers [24, 25, 26], including MRS. Available MRS studies in ALS are mainly focus on motor areas and report reduced NAA in regions spanned by the pyramidal tract while few extra-motor MRS studies provide evidence of metabolic changes in prefrontal regions and subcortical structures . However, the profile of neurometabolic changes is relatively under-evaluated in other MND phenotypes, such as primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), spinal bulbar muscular atrophy (SBMA) or Kennedy disease, spinal muscular atrophy (SMA). Moreover, more than 50% of ALS show cognitive impairment [28, 29, 30, 31] which is not restricted to traditional frontal-related executive dysfunction [32, 33, 34] and corroborates with structural and functional alterations in cross-sectional and longitudinal MRI studies [7, 35, 36, 37, 38, 39, 40, 41, 42, 43]. In addition, other MND phenotypes present variable changes across different cognitive functions [44, 45] in line with neuroimaging findings [46, 47, 48, 49, 50, 51]. However, neurometabolic changes in extra-motor regions are only seldom examined and mainly in ALS .
Accordingly, the aim of this review is to systematically review the current literature of MR spectroscopy studies in MNDs, highlight clinical implications compared and/or in addition to gold standard techniques, identify pertinent gaps in the literature, evaluate the clinical and academic role of MR Spectroscopy and outline future directions.
A formal literature review was conducted on PubMed between September 2021 and
January 2022 in accordance with the PRISMA 2020 guidelines. Two reviewers (F.C.
and P.B.) conducted the search and data extraction independently. The search term
“spectroscopy” and “spectroscopic” was paired individually with the following
keywords: “amyotrophic lateral sclerosis”, “motor neuron disease”, “PLS”,
“SBMA”, “Kennedy’s disease”, “PMA”, “SMA”, “ALS-FTD” (Fig. 2).
Selection criteria included original research articles pertaining to spectroscopy
brain studies, published during the past three decades. Only human studies were
reviewed. Only articles in English were selected for review. Opinion pieces,
previous review articles, case reports, research studies without imaging were not
reviewed. The reference list of identified papers were also reviewed for articles
that might have been missed. We developed an MS EXCEL-type database including
specific fields based on which we comprehensively and quantitatively reviewed all
studies. The following fields/variables were included for data extraction: first
author; year of publication; study sample size; patients’ sample size; healthy
controls’ sample size; patients’ and healthy controls’ age (mean
Systematic review flow chart.
Based on the above search strategy, a total of eighty-seven original research studies were identified. Forty-one studies were published after 2010 (Supplementary Table 1), thirty-two studies between 2001–2009 (Supplementary Table 2) and fourteen studies before 2000 (Supplementary Table 3).
The majority of the studies are cross-sectional (62/87 studies) whereas among the 25 longitudinal studies, 10 studies examine pre- and post-treatment metabolic changes, i.e., riluzole [10, 52, 53], gabapentin [53, 54], creatine supplementation [55, 56], brain-derived neurotrophic factor (BDNF) , olfactory ensheathing cells transplantation , minocycline , growth hormone , and stem cell .
Most of the studies have recruited patients with definite, probable, possible, and clinically probable laboratory supported ALS. The total number of ALS patients included in these studies ranges from n = 1  to n = 154 . Currently, the largest total number of ALS patients in a single-centre study is n = 154  compared to a multicentre study including as many as 74 ALS patients . Only few studies include non-ALS MND groups, i.e., PLS [65, 66, 67, 68, 69, 70], PMA [66, 69, 71, 72, 73], SBMA (Kennedy disease) [74, 75, 76], presymptomatic SOD1+ individuals . The total number of PLS patients ranges from n = 6 [66, 69] to n = 18 , the total number of PMA ranges from n = 2  to n = 9 [66, 69], the total number of SBMA (Kennedy disease) ranges from n = 5  to n = 10  while the only presymptomatic study includes 24 SOD1+ individuals . Across the 87 studies, 45 studies have a disease sample size between 1–15 patients, 27 studies have a disease sample size between 16–30 patients, nine studies have a disease sample size between 31–50 patients and six studies have a disease sample size larger than 50 patients.
In the majority of studies, the ALS patients are contrasted to demographically-matched healthy individuals. Ten studies do not include a healthy control group [10, 15, 56, 59, 60, 61]. Six studies include a disease-control group other than MND, i.e., MS, chronic polyneuropathy, and acute polyneuropathy , patients not meeting the El Escorial criteria , Alzheimer’s disease , other neurological diseases or various neurological diseases restricted to peripheral nerves .
Four studies do not directly report participants’ age [15, 63, 68, 80]. Mean patients’ age ranges from 45.75 years  to 67.7 years . One study includes only male patients . A comprehensive clinical characterisation of study participants’ clinical profiles is important to develop meaningful imaging markers. The focus of clinical variables varies among the identified studies. The disease duration profile of patients enrolled in the studies varies significantly ranging considerably even within the same study, e.g., 0–82 months  while 17 studies do not directly report patients’ disease duration [14, 18, 53, 55, 61, 63, 68, 80, 84, 85, 86, 87, 88, 89, 90, 91, 92]. Age onset is reported in eight studies [15, 67, 74, 75, 76, 93, 94, 95]. Most studies (57/87) focus on motor-related disease severity, mainly measured using ALSFRS or ALSFRS-R, but five out these 57 studies do not provide any quantitative data for disease severity [11, 52, 84, 89, 96]. Onset type (e.g., bulbar and spinal symptoms) is reported in 43 studies [12, 15, 56, 58, 59, 60, 61, 64, 67, 70, 71, 77, 79, 81, 83, 84, 89, 93, 94, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120]. Progression rate is reported in 13 studies [64, 65, 70, 82, 94, 97, 101, 103, 107, 121, 122, 123, 124]. Riluzole treatment is reported in 15 studies [15, 52, 56, 59, 60, 93, 99, 102, 108, 117, 121, 125, 126, 127, 128]. Respiratory status (i.e., forced vital capacity, slow vital capacity, non-invasive ventilation) is only available in 19 studies [11, 52, 54, 61, 65, 66, 69, 97, 98, 99, 101, 105, 106, 111, 117, 122, 125, 128, 129]. Tapping rate is measured in eight studies (only finger [67, 90, 103, 123], both finger and foot [52, 65, 111, 115]), yet in one study data are not reported . Additional cognitive status is only rarely reported. Interestingly, only 11 studies include cognitive scales or tests, i.e., mental status/cognitive screening: Mini-Mental State Examination (MMSE) [62, 71, 82, 111, 117], Addenbrooke’s Cognitive Examination (ACE) , Edinburgh Cognitive and Behavioural ALS Screen (ECAS) [64, 93, 121]; verbal fluency [71, 98, 109, 120]; executive functions: Wisconsin Card Sorting Test (WCST) [62, 117, 120]; memory tests: Rey Auditory Verbal Learning Test, Recognition Memory Test, Consonant trigrams test [62, 120]; perceptual and constructional dexterities: Motor Free Visual Perception Test , Block Design [62, 120]; expressive language: Cookie Theft - Boston Diagnostic Aphasia Examination ; behavioural inventories [62, 120]. In addition, one study includes cognitively preserved patients  and another study provides patients’ cognitive status (preserved vs impaired)  but additional information regarding patients’ cognitive evaluation is not provided.
Except from the three whole-brain studies which enable the identification of metabolic changes in both motor and extra-motor regions [83, 85, 107], almost all single and multi-voxel studies focus on motor-related regions (precentral gyrus, supplementary motor area, premotor cortex, postcentral gyrus, subcortical motor related WM areas (corpus callosum, coronal radiata, internal capsule, cerebral and cerebellar peduncles)). Extra-motor regions are examined in 25 single- or multi-voxel studies [12, 52, 53, 54, 56, 62, 64, 71, 73, 87, 88, 89, 90, 98, 101, 109, 111, 112, 117, 120, 123, 126, 129, 130, 131]; interestingly, only five of them are dedicated extra-motor studies but mostly examine frontal areas [56, 71, 98, 109]. Basal ganglia and thalamus are examined in one multi-voxel study , brainstem in 14 studies [53, 59, 67, 74, 75, 76, 80, 101, 102, 112, 121, 127, 132, 133] and cerebellum in one single-voxel study . On the other hand, there are three studies focusing on the cervical cord [11, 106, 110].
None of the studies report genetic screening for the GGGGCC hexanucleotide repeat expansions. Two studies use genetic testing for SOD1 [11, 72], one study reports genetic screening for six familial ALS included in the study  while one study provides data for TSPO Ala147Thr polymorphism based on 11C-PET (11C-PBR28) . Genetic analysis for the number of CAG repeats is finally reported in three studies including patients with SBMA (Kennedy disease) [74, 75, 76].
Twenty-three studies include additional imaging modalities. Specifically, 17
studies use DTI [66, 69, 77, 78, 81, 82, 84, 93, 94, 97, 101, 107, 113, 114, 124]
or DWI [62, 70], seven studies provide signal intensity changes [68, 78, 81, 95, 112, 119, 124, 130], four studies report GM volume or thickness changes [68, 97, 119, 131], one study uses resting state fMRI , one study uses T1
Forty-five studies use 1.5 T (four are published between 2011–2016; [61, 84, 106]), one study uses 2 T , thirty-seven studies use 3 T (among which one is
published before 2000; ), one study uses 4 T , one study uses 3 T and 7
T  and two studies use 7 T [121, 122]. Forty-nine studies apply a
single-voxel technique, 23 studies apply a multi-voxel technique, one study uses
both single-voxel and multi-voxel techniques while four studies apply whole-brain
spectroscopy. Two studies use diffusion tensor spectroscopy  and diffusion
weighted spectroscopy  while one study uses cranial phosphorus-31 MRS
(31P-MRS; ) instead of
18.104.22.168 Cortical Motor Regions
MR Spectroscopy studies verify motor abnormalities in ALS based on the pattern of metabolic changes in primary motor cortex/precentral gyrus and other motor regions, including the supplementary motor area (SMA). Several studies provide evidence of reduced NAA [66, 72, 88, 99, 119, 122, 127], tNAA , NAA/Cr [63, 68, 91, 94, 95, 96, 99, 103, 112, 113, 115, 116, 117], NAA/tCr [66, 105], NAA/Cho [91, 92, 95, 99, 113, 115, 116, 123, 131], NAA/Glx , NAA/Cho+Cr [90, 95], NAA/mIns [99, 102, 115], tNAA/Cr , tNAA/mIns . Interestingly, abnormal NAA/Cr ratio is found in most patients in the largest single-centre study which also reports lower NAA/Cr ratio in patients with definite UMN signs compared to patients with no UMN signs . Precentral gyrus reduced NAA/Cr and NAA/Cho is found both in affected and non-affected hemispheres of ALS patients when compared to healthy individuals while NAA/Cho is significantly lower in the affected compared to non-affected hemisphere within ALS . Other relatively common metabolic changes in primary motor cortex include increased mIns [99, 127], mIns/NAA , mIns/Cr [99, 115] and mIns/tCr . Increased mIns/Cr ratio is reported to ALS patients with UMN involvement compared to patients with LMN involvement . Lower NAA/Cr ratio is associated with lower FA and reduced 11C-PBR28 in bilateral precentral gyri while higher mIns/Cr is associated with lower FA and reduced 11C-PBR28 in bilateral precentral gyri . Two whole-brain studies identify reduced NAA/Cr in right precentral gyrus  and simultaneous decreased NAA and increased Cho in motor and premotor areas extending along motor fibers based on multivariate but not univariate analysis . Reduced GABA is also demonstrated in two studies [108, 127].
The NAA/Cho ratio in primary motor cortex is found to have the highest sensitivity and specificity compared to other ratios (NAA/Cr and Cho/Cr) in discriminating ALS from healthy individuals . The combination of NAA/Cho+Cr ratio and hypointensities in precentral gyrus and hyperintensities in the corticospinal tract have a diagnostic accuracy of 79.4% compared to the diagnostic accuracy of 72.3% for the precentral gyrus NAA/Cho+Cr alone . By comparing the MR Spectroscopy changes with classic MRI metrics, Chan and colleagues (1999) report a sensitivity of 73% for motor cortex NAA/Cr (vs 38% for corticospinal tract hyperintensities and 23% for widening of central sulcus) in ALS, 88% (vs 12% for corticospinal tract hyperintensities and 12% for widening of central sulcus) in ALS with probable UMN signs, 67% (vs 10% for corticospinal tract hyperintensities and 14% for widening of central sulcus) in PLS and 17% (vs 0% for corticospinal tract hyperintensities and 38% for widening of central sulcus) in non-UMN disorders .
Two multimodal studies provide interesting findings with regards to the pattern of motor metabolic changes in different El Escorial groups (i.e., definite, probable, possible, suspected). The NAA/Cho in primary motor cortex shows variable percentage of changes across El Escorial groups and compared to TMS, MR Spectroscopy detects more patients with UMN involvement in the suspected group while the TMS detects more patients with UMN involvement in possible, probable and definite El Escorial groups . According to a more recent single-voxel MR Spectroscopy and DTI study, it appears that increased mIns/Cho and Cho/Cr ratios in precentral regions are found in all ALS subgroups (definite/probable, possible/suspected, suspected) compared to healthy individuals while reduced NAA/Cr is found only in definite/probable ALS patients .
One of the most recent studies included in this review combines MR Spectroscopy, DTI and resting-state fMRI to examine SMA abnormalities in 25 ALS patients; patients show increased Glu/NAA and Glu/GABA compared to healthy individuals but no differences in absolute values (Glu, NAA, GABA), fractional anisotropy (FA) or regional homogeneity (ReHo) of the SMA .
22.214.171.124 Cortical Extra-Motor Regions
Most studies focus and/or report changes in NAA absolute values or ratios relative to healthy individuals. Except for three studies that do not identify any frontal-related metabolic changes [87, 88, 90], changes are evident in medial prefrontal cortex (reduced NAA/Cho: ; reduced NAA/mIns: ), mid-cingulate cortex (reduced NAA/Cho: ) and posterior superior frontal gyrus (reduced NAA/Cr: ). In a longitudinal study where bulbar-onset and limb-onset ALS patients were compared to healthy individuals, baseline reduced NAA/Cr is reported in ACC in bulbar-onset ALS but not in limb-onset ALS but six months after the first MRI scan, reduced NAA/Cr is also found in limb-onset ALS patients . Atassi et al.  and colleagues examine the metabolic profile after different dosages of creatine supplementation and report increased Cr and Cr/Cho and decreased Glx in the frontal cortex between baseline (pre-treatment) and the higher dosage of creatine. Parietal regions are seldom examined and most cross-sectional [89, 90, 117] and longitudinal [52, 54, 87, 129] studies fail to identify any changes, with the only exception been the pioneer MR Spectroscopy study by Pioro et al.  and colleagues where decreased NAA/Cr is found in superior parietal gyrus/precuneus, although to a lesser extent than in primary motor cortex. On the other hand, even though several single or multi-voxel MR spectroscopy studies often include occipital regions as a reference region and do not find significant metabolic changes [53, 71, 88, 89, 112, 130, 131], a whole-brain cross-sectional study in 16 ALS patients and 10 healthy individuals demonstrates reduced NAA/Cr and NAA/Cho not only in inferior frontal operculum, caudate, Hesch gyrus, and supramarginal gyrus but also in several occipital regions (lingual gyrus, superior occipital lobe, inferior occipital lobe, cuneus) . Interestingly, none of the available studies examine metabolic changes in temporal regions.
126.96.36.199 Subcortical Structures
There are only few available but inconsistent data for the metabolic profile of subcortical structures [85, 111, 123]. A 3 T study dedicated to basal ganglia and thalamus demonstrates reduced NAA and NAA/Cho and increased Cho in lentiform nucleus and thalamus but not in the caudate in ALS compared to HC ; however, a 1.5 T study failed to reproduce these findings in the thalamus . Another whole-brain study provides evidence of reduced NAA/Cho, NAA/Cr in left caudate but does not identify any metabolic changes in the thalamus .
Most studies indicate reduced NAA or NAA/Cr ratios in brainstem [53, 102, 127, 132, 133]. Other studies failed to identify metabolic changes in the NAA ratios (NAA/Cr, NAA/Cho) of the brainstem . Increased Glx/PCr ratio is reported in one study , yet some others failed to identify changes in Glu or Glx . Of note, findings from a recent longitudinal study indicate increased Glx .
Only one study specifically focuses on the cerebellum ; however, no significant differences were identified in Cho, Cr/PCr and NAA.
188.8.131.52 White Matter
Among the studies which examine metabolic changes in white matter, most of them focus in the motor WM pathways and the corticospinal tract [15, 58, 62, 67, 70, 81, 83, 84, 86, 87, 88, 90, 96, 107, 108, 113, 127, 128], two studies examine the corpus callosum [84, 93] and one study examines frontal WM . One of the most consistent finding is reduced NAA and/or NAA/Cr in subcortical motor WM , posterior limbs of the internal capsule , corona radiata , centrum semiovalis , cerebral peduncle and internal capsule , and CST [107, 128]. Some studies that examine metabolic changes throughout the entire CST  or combined parts of it [81, 90] and different segments find reduced NAA throughout the course of the CST  or the combined segments  but not on every CST segment [90, 128]. Significant metabolic alterations have also been reported in motor and premotor areas extending along motor fibers (decreased NAA and increased Cho) using unbiased whole-brain MR spectroscopy . Reduced NAA/Cr and NAA/Cho in CST appear to be found not only in definite and probable but also in possible ALS relative to healthy individuals . Interestingly, a multimodal MR Spectroscopy and DTI study indicates reduced NAA throughout the course of the CST and reduced fractional anisotropy (FA) and increased mean diffusivity (MD) but highlights the greater anatomical specificity compared to FA and MD . Other metabolic changes include higher mIns in subcortical motor WM , increased Cho in the entire CST, increased Cho/NAA in the entire CST and different segments , increased Glx/Cr and Glu/Cr  and Cho  in posterior limbs of internal capsule, reduced Glx/Cr in cerebral peduncle and internal capsule and reduced Cho/Cr in internal capsule . Absence of metabolic changes including NAA are more rarely reported [88, 108]. On the other hand, reduced NAA/Cr and NAA/Cho in definite and probable ALS but not in possible compared to healthy individuals has been reported in the splenium of the corpus callosum . A more recent study does not reproduce these findings in the corpus callosum using DTI-MRS but identifies higher D(tNAA) variability in ALS .
184.108.40.206 Spinal Cord
The few MR Spectroscopy studies indicate reduced metabolic ratios in ALS compared to healthy individuals: NAA/Cr [11, 106, 110], NAA/Cho , NAA/mIns [11, 106, 110], mIns/Cr [11, 106] and Cho/Cr .
Among the 25 longitudinal studies, 14 studies examine neurometabolic changes in ALS over varying time intervals [15, 18, 65, 69, 78, 86, 87, 100, 102, 118, 120, 121, 130, 134] and all but five [64, 86, 87, 120, 130] focus exclusively on motor regions. Earlier studies provide evidence of progressive decline in NAA  and NAA/Cho [118, 134] with the highest changes being observed in patients with normal baseline spectroscopy data . In a longitudinal study (0 m, 3 m, 6 m) of metabolic changes in GM motor region and subcortical paraventricular WM including the corticospinal tract, there is a progressive reduction of the NAA (0 m vs. 3 m) for the clinically less affected hemisphere and of NAA (0 m vs. 6 m) for both the less and more affected hemisphere with regards to the GM motor region but not the WM . In a recent study where ALS patients are subdivided into fast and slow “progressors” and those with UMN and LMN involvement, it appears that progressive decline in NAA/Cr and NAA/Cho is preferentially detected in “fast progressors” as well as patients with predominant UMN involvement . A previous comparative study of ALS and AD where motor cortex and mesial temporal lobe is examined in each group, respectively, finds a general trend for further reduction in motor cortex NAA/Cho over time but inter-subject variability in ALS and a trend towards progressive reduction of mesial temporal lobe NAA/tCr and NAA/Cho in AD . However, the study does not examine mesial temporal lobe longitudinal changes in ALS. Few studies have examined longitudinal alterations in association with varying levels of diagnostic certainty (definite, probable, possible, suspected ALS) but yield inconsistent findings [78, 86, 87]. Interestingly, among the studies which examine both motor and extra-motor longitudinal changes in ALS, there are inconsistent findings with some studies reporting no prefrontal changes , other studies showing reduced in NAA at 9 m but not earlier at 3 m in probable/definite but not in possible/suspected ALS  and some other studies identifying reduced NAA/Cr both in motor cortex and anterior cingulate cortex in limb- but not in bulbar-onset ALS .
In addition, there are 11 studies which evaluate specific treatment-related neurometabolic changes [10, 52, 53, 54, 55, 56, 58, 59, 60, 61, 129]. In treatment studies of riluzole, there is evidence of increased NAA/Cr ratio in motor cortex [10, 52] and SMA . Two studies examine the neurometabolic changes after creatine supplementation in motor cortex  and frontal cortex . They show post-creatine supplementation decline in NAA/tCr, NAA/Cho, Glx/tCr and Glx/Cho ratios in healthy individuals and decline in Glx/tCr and Glx/Cho but no changes in NAA/tCr (which was reduced at baseline relative to healthy individuals) and NAA/Cho ratios in ALS . It also appears that there is a significant Cr increment, Glx decline and Cr/Cho increment in the frontal cortex between the pre-treatment and the highest dosage of creatine supplementation . Longitudinal studies examining the effect of stem-cells , growth-hormone , minocycline , gabapentin  do not find any significant post-treatment differences compared to the baseline metabolic profile of ALS.
There are only few MR Spectroscopy studies that have recruited patients with PMA. Among the five of them which are included in the present review [66, 69, 71, 72, 73], only four provide a direct comparison between PMA and HC [66, 72, 73]. Two earlier studies do not find any metabolic changes neither in the motor cortex [72, 73] nor in sensory and parietal regions  or the cerebellum ; however, more recent studies report decreased NAA/tCr ratio but not NAA in PMA compared to HC [66, 69]. In another study where metabolic changes are examined in dorsolateral prefrontal cortex (DLPFC), a supplementary analysis in a subgroup of patients with PMA shows higher NAA/Cr in left DLPFC and average DLPFC compared to a group of definite, probable and possible ALS .
All available data on PLS are related to motor regions. There is consistent evidence for reduced NAA/Cr ratios in PLS compared to healthy individuals [65, 66, 67, 68, 69] and to advanced-phase ALS . One study also reports increased mIns compared to healthy individuals .
The few MRS studies in SBMA (Kennedy disease) indicate inconsistent findings [74, 75, 76]. Two long echo-time MR spectroscopy studies demonstrate reduced NAA/Cr in motor cortex [74, 76] and NAA/Cho in motor cortex and brainstem . A short echo-time study fails to reproduce these findings in motor cortex where only increased mIns was found .
Reduced mIns/Cr, NAA/Cr and NAA/mIns in the cervical cord (C1-C2) is reported in the only study including presymptomatic cases compared to healthy individuals .
Even though there are few studies with available post-mortem data, none of them provide any direct correlation with MRS findings.
Correlations between disease duration and metabolic ratios in motor-related GM and WM regions are found in number [103, 104, 112, 113, 118, 127] but not all [10, 52, 70, 74, 76, 77, 94, 95, 105, 107, 115, 116, 124] studies. Moreover, there are conflicting findings, i.e., positive association with motor cortex NAA/Cr  and negative association with right precentral gyrus NAA/Cr , positive association with precentral NAA/Cho  and negative association with primary motor cortex [112, 118] and medulla oblongata  NAA/Cho. Lower NAA/Glu  and GABA  in motor cortex and higher mIns in subcortical WM caudal to the motor cortex  are also found to be associated with longer disease duration.
Studies that examine the association between metabolic changes in MND and disease severity (mostly measured through ALSFRS or ALSFRS-R) or progression rate provide inconsistent findings (Supplementary Table 1) and there are several studies that fail to establish any relation [10, 52, 71, 76, 77, 94, 95, 96, 103, 105, 110, 113, 115, 116].
Positive correlations between disease severity and metabolic profile of motor regions (GM and/or WM) is found in several studies (NAA: [66, 69, 107, 112, 127]; NAAx: ; tNAA/Cr: [102, 126]; NAA/Cr: [99, 101, 114]; NAA/Cho (caudate): ; NAA/Glx: ; NAA/mIns: ; tNAA/tmIns: ; Glu ; mIns: [78, 102]; NAA/Cr+PCr: ; Cho/Cr+PCr: . On the other hand, negative association are also reported with motor regions mIns [78, 99], Glx , mIns/Cr , NAA/Cho , Glx/PCr . In addition, a negative association is reported for brainstem NAA/Cr ratio and the severity of brainstem signs and symptoms . The only 31P-MRS study in ALS also reports a negative association between brainstem phosphocreatine and ADP and disease severity measured through ALSFRS-R . Higher NAA/Cr and NAA/mIns in the cervical cord of ALS patients is associated with higher ALSFRS-R and non-bulbar ALSFRS-R and lower annual decline of ALSFRS-R and non-bulbar ALSFRS-R . In a longitudinal study where olfactory ensheathing cell transplantation was applied in seven patients who underwent MR Spectroscopic examination focused on motor regions (cerebral peduncle, genu and posterior limbs of internal capsule, corona radiata, precentral gyrus), higher NAA/Cr was found in certain anatomical positions which confirm the neurological and EMG findings in two cases with ALSFRS improvement following the transplantation . In another longitudinal study with stem-cell transplantation, baseline (M0) NAA/Cr ratios in the precentral gyrus at the dominant and non-dominant hemispheres are found to be associated with follow-up (M12) ALSFRS-R .
Only few studies establish an association between disease progression rate and metabolic changes. Disease progression rate is negatively associated with motor cortex NAA [89, 97], tNAA/tCr  and NAA/Cho [113, 123]. In a recent longitudinal study where ALS patients are categorized as fast and slow progressors, motor cortex NAA/Cr ratio is lower in fast compared to slow progressors patients at baseline and further decline in motor cortex NAA/Cr and NAA/Cho ratios is reported in fast progressors ALS .
Few studies examine differences in metabolic profile between different onset-type phenotypes. Bulbar-onset ALS patients show reduced motor cortex NAA [99, 119] and NAA/Cr+PCr  compared to limb-onset patients in another diffusion tensor spectroscopy study, limb-onset patients show a more widespread pattern of alterations characterized by increased MD tCr and MD tCho, AD tCho, RD tCr and RD tCho, and decreased tNAA/tCr and tNAA/tCho in precentral gyrus compared to healthy individuals while bulbar-onset patients show only increased MD tNAA and tNAA/tCho . However, there are studies that fail to establish any association between onset type and a different pattern of metabolic alterations in motor regions [52, 108, 118] or dorsolateral prefrontal cortex .
Finger and foot tapping rate are used as simple but objective measurements of UMN function in MND and other clinical groups with UMN deficits that show reduced rate to produce rapid and repetitive movements of the hands and feet. Some studies report positive correlations between finger tapping rate and motor cortex NAA [66, 69], NAAx  and NAA/Cr [67, 103], as well as between foot tapping rate and NAA/tCr . Negative correlations are noted between finger tapping rate and NAA/Cho in mid-cingulate cortex , Cho/NAA in the precentral gyrus part of the corticospinal tract and centrum semiovalis  and mIns in motor cortex , as well as between foot tapping rate and Cho/NAA in posterior limbs of the internal capsule . Other studies do not find any association .
Only few reports are available regarding the predictive role of metabolic changes in motor and extra-motor regions and patients’ survival. Primary motor cortex NAA/Cho ratio, older age and shorter symptom duration are found to be associated with reduced survival in a previous ALS study . In a more recent study, NAA/mIns in the medial prefrontal cortex is reported as an independent predictor of survical in ALS patients two but not five years after the MRI scan in addition to age, ALSFRS-R and forced vital capacity while the same metabolic ratio is also identified as the only significant predictor of survival in a multivariate Cox regression model .
As cognitive measures are only seldom used and extra-motor regions are rarely examined in the MR Spectroscopy studies, there are only scarce reports of the association between cognitive processes and metabolic changes in MND [64, 71, 109, 117]. Among the available MR Spectroscopy studies with cognitive measures, some studies report a positive association between NAA/Cr in frontal/prefrontal areas and performance in executive measures [71, 117] and identify reduced NAA/Cho in cognitively-impaired ALS compared to HC but not compared to cognitively-unimpaired ALS . Patients with ALS and impaired letter fluency show reduced left and average dorsolateral prefrontal cortex NAA/Cr compared to ALS patients with normal letter fluency, even after excluding a subgroup of PMA . There is no reported correlation between non-executive measures (i.e., language, memory, perception, constructive dexterities) and metabolic changes in MND groups.
While structural, diffusivity and functional methods are often applied in conjunction in MND , spectroscopy is often performed in isolation. Only a minority of the neuroimaging community in MND would routinely use MRS techniques, which has led to strikingly divergent research strategies in structural and spectroscopic imaging. While MRS studies pioneered innovative designs, such as monitoring drug response [10, 52, 54, 129], evaluation of cord metabolites , assessment of changes in non-ALS MNDs [74, 75, 76], structural studies turned their attention to frontotemporal regions , infratentorial changes , extra-pyramidal deficits , and machine-learning applications [139, 140] relatively earlier. The relevance therefore of reviewing advances in MRS in MND points beyond metabolic imaging, as it offers valuable lessons for non-spectroscopic imaging in MND.
The review of published MRS studies reveals diverse methodological approaches. The majority of MRS studies adopt a single-voxel approach and only few capitalise on recent advances in whole-brain spectroscopy [85, 107]). With the exception of a 4 Tesla  and three 7 Tesla study [102, 121, 122], most studies relied on 1.5 or 3.0 Tesla field strength. The vast majority of studies applied 1H-MRS and only few utilised MRSI [10, 52, 54, 60, 66, 67, 70, 71, 73, 83, 84, 86, 87, 88, 90, 95, 97, 103, 113, 114, 116, 123], diffusion-tensor spectroscopy [93, 100], or 31P-MRS . With the exception of two superior spinal cord studies [11, 106, 110] all studies focused on the evaluation of cerebral metabolites. The most commonly evaluated brain region was the motor cortex, but targeted brainstem [80, 101, 102, 121, 132, 133] and innovative thalamus studies were also identified [13, 123]. Strikingly few studies have evaluated fronto-temporal regions [71, 86], and only few studies acquired spectra from parietal or occipital regions [12, 89, 90, 126, 131] often as ‘reference voxels’. A potential limitation of our study is that search and data extraction was conducted on a single database. Methodological considerations have also been comprehensive reviewed in other clinical groups .
The majority of MRS studies in ALS report composite functional disability scores (ALSFRS) and some pursue direct correlation analyses between cerebral metabolite ratios and ALSFRS even though ALSFRS is also affected by anterior horn degeneration. To delineate the clinical impact of UMN degeneration some studies record finger tapping rates [103, 123], foot tapping [52, 142], ‘rapid bulbar muscle movements’ , UMN scores , which allow for more accurate correlation analyses. Interestingly, the presence of pseudobulbar affect (PBA) or a positive jaw jerk is not commonly reported despite their association with corticobulbar dysfunction [143, 144]. Site of onset (bulbar/spinal) is often reported albeit the relevance of onset may be relatively limited by the time spectra are acquired . While site of onset is invariably reported, side of onset (left or right) is seldom noted, despite its relevance to the laterality and symmetry of findings. Progression rates, clinical staging, respiratory function profiles, Riluzole therapy are commonly reported among core clinical variables even if clinico-metabolic associations are not sought. In striking contrast to structural studies, accompanying cognitive and behavioural information is scarce in published MRS reports. ECAS [64, 93, 121], letter fluency [71, 98, 109] or MMSE [71, 82, 117] is sometimes reported, but detailed neuropsychological testing is seldom available [62, 120]. Despite their clinical relevance [146, 147] accompanying behavioural data , tests of social cognition and apathy are seldom appraised in MRS studiers despite their association with specific brain regions.
The review of published MRS studies also reveals common study limitations. Most of the published MRS studies in ALS, particularly earlier studies in the field, only report MRS alterations and corresponding structural, diffusivity and functional changes are not evaluated. More recent studies commonly describe associations with diffusivity changes, typically with fractional anisotropy (FA) [77, 93, 94, 107]. Some studies acquire complementary functional data, such as rsfMRI , PET  or TMS [66, 67, 92, 93] correlates. The lack of accompanying post mortem assessments makes the histological interpretation of metabolic changes challenging. Only three studies [63, 66, 82] described pathological correlates of ante mortem MRS findings. One of the most notable limitations identified is the lack of MRS studies in GGGGCC hexanucleotide repeat carriers in C9orf72. While the structural correlates of C9orf72 have been extensively investigated [137, 148, 149] the metabolic correlates of this genotype remains to be characterised. With the exception of a few SOD1 studies [11, 72], and a few studies in genetically-confirmed SBMA (Kennedy’s disease) [74, 75, 76] the vast majority of existing MRS studies did not stratify their cohort by genetic profile. This is a missed opportunity at a time when genotype-associated clinical traits, differences in survival, disability profiles are well characterised, and emerging therapies such as antisense oligonucleotides (ASO) target specific genotypes. A notable shortcoming of existing studies is the overwhelming focus on ALS. MNDs encompass a multitude of clinically diverse phenotypes with differences in progression rates , survival , non-neurological involvement , spinal cord pathology . The vast majority of existing MRS studies only evaluate patients with ALS, and only a few investigated changes in PLS [65, 66, 68], PMA [66, 69, 72], or SBMA [74, 75, 76]. PLS is associated with longer survival than ALS  and is a difficult to diagnosis to establish early in the course of the disease  as UMN-predominant ALS may present similarly despite running a more sinister course. There is a lack of spectroscopy studies in rare MNDs such as post-polio syndrome (PPS) where progressive changes are sometimes detected decades after the initial cord insult . The characterisation of distinguishing metabolic signatures in non-ALS MNDs should therefore be an important priority of future research. Less than a third of published MRS studies are longitudinal, and the majority of these studies have a mere two-time point design which preclude the assessment of non-linear changes, ceiling and flooring effects. MNDs, ALS in particular, are rapidly and inexorably progressive neurodegenerative conditions, where cross-sectional studies merely offer a snapshot of metabolic change and patients are often in a different stage of their disease trajectory . In cross-sectional studies, the “disease-duration” or “symptom duration” profile of participants is often not reported despite being a key determinant of the detection and magnitude of pathological change. When this information is reported, it is often apparent that patients with a considerable symptom duration range have been included which makes the interpretation of findings difficult. Reporting disease duration is indispensable, as for example the absence of MRS findings may be due to early-stage disease or technical factors. The admixing of patients with very different disease duration may be a necessity due to cohort size limitations, but the impact of disease duration should always be carefully considered. A requisite of future MRS studies therefore is a prospective, multi-timepoint longitudinal design, with a relatively homogenous symptom duration profile at enrolment and uniform follow-up intervals. While presymptomatic structural and diffusivity changes have been consistently described in ALS [156, 157], there is also a striking scarcity of metabolic studies of asymptomatic mutation carriers . It is currently unclear if early metabolic changes may precede structural degeneration and augur focal vulnerability before structural neurodegeneration ensues. If that is the case, MRS may be particularly useful to detect early pathophysiological changes, which may have ramification for recruitment in pharmacological trials. The nuanced characterisation of metabolic alterations in asymptomatic mutation carriers may help to elucidate anatomical propagation patterns  and may have a prognostic role in predicting age of symptom onset. The challenges of recruiting asymptomatic mutations carriers are well known, but viable strategies have been developed to acquire large multi-site datasets and meaningfully interpret premanifest radiological changes [156, 159, 160].
On the review of studies by date of publication, cohesive methodological trends emerge: (1) with ever improving scanner designs, studies increasingly rely on higher field-strengths, (2) with the recognition of extra-motor manifestation in ALS, studies increasing evaluate non-motor regions, (3) with the availability of whole-brain techniques multiple brain regions are now often simultaneously evaluated [85, 107], (4) with the availability of novel pulse sequences, MRS data is increasingly complemented by simultaneously acquired structural, functional or PET data which aid the interpretation of metabolic changes (5) with the availability of open-source software there is a shift from reliance on in-house pipelines to validated robust analysis suites which helps the comparison and replication of research findings.
Existing MRS studies in MND indicate that MRS has the potential to define disease associated (PLS, SBMA, ALS) metabolic signatures and capture progressive changes over time. Even though the majority of ALS are sporadic cases with no clear genetic linkage, there is a considerable 10% of cases with familial inheritance  and there are collective efforts to study presymptomatic ALS cases and identify biomarkers [162, 163, 164]. With the advent of whole-brain protocols MRS could be utilised to clarify anatomical propagation patterns from the presymptomatic phase, through the peri-diagnostic stage, well into the late phases of the disease and help to establish if metabolic alterations precede, coincide or follow structural and diffusivity changes. Multi-time point metabolic profiles may meaningfully contribute to the corticofugal versus corticopetal debate, and offer validation opportunities for proposed histopathological staging systems in vivo [165, 166]. Animal imaging studies in MND rarely implement MRS despite the high-field strength generated by small-bore scanners and the opportunity to link specific spectral profiles to histopathological changes.
The insights gained from MRS studies in MND to date have not been translated into practical clinical applications. Given the differences of spectra between patients and controls, the early diagnostic utility of spectroscopy in suspected MND cases and the potential role in distinguishing MND phenotypes have not been fully explored. ROC curves and various machine-learning frameworks  in structural imaging suggest that imaging may have a role in delineating MND phenotypes  or distinguish early-stage ALS patients from healthy individuals  with adequate accuracy. The clinical utility of MRS should not only be examined form a diagnostic standpoint, but its potential to track longitudinal changes and be used as a putative monitoring marker in clinical trials should also be explored. Finally, similar to structural imaging [169, 170], the prognostic correlates of metabolic alterations should be carefully evaluated.
MRS needs to be integrated into multimodal imaging protocols to establish its comparative detection sensitivity, relative advantages and its diagnostic, monitoring, and predictive potential. Future studies should address whether MRS can be developed into a viable clinical tool to differentiate various phenotypes, monitor progression, and detect response to therapy. The practical demands of clinical imaging require relatively short acquisition times, transparent and reliable data interpretation pipelines, uniformity of voxel placement and ease of harmonising protocols across clinical sites. There is a relative urgency to shift the emphasis from descriptive, MRS-only academic studies to the development of protocols with practical clinical utility.
MRS remains one of the most promising MR-based imaging modalities in MND, which offers invaluable metabolic data non-invasively. Existing studies have already cemented the academic role of MRS by characterising neurodegenerative processes in MND. An urgent priority of future studies is to define, gauge, and establish the clinical role MRS in expediting the diagnosis, monitoring disease progression and assessing response to therapy.
The manuscript was drafted by FC, EK, GDA, IS, and PB. EK, GDA and IS contributed to the MRS analyses and the results presented in Fig. 1. The final draft was reviewed for intellectual content by GV, VZ, AM, IE, and MR.
The acquired spectra presented in Fig. 1 was obtained as part of a research MRS
study which was approved by the local ethical committee of Aeginition Hospital;
National and Kapodistrian Univeristy of Athens; GR
We would like to thank the participants of the research MRS study based on which we generated Fig. 1.
Foteini Christidi is supported by the EU-IKY Scholarship Program (European Social Fund-ESF), the Greek “Reinforcement of Postdoctoral Researchers” grant (5033021) of the “Human Resources Development Program, Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF 2014-2020). Professor Peter Bede is supported by the Health Research Board (HRB EIA-2017-019 & JPND-Cofund-2-2019-1), the Irish Institute of Clinical Neuroscience (IICN), the Spastic Paraplegia Foundation (SPF), the EU Joint Programme – Neurodegenerative Disease Research (JPND), the Andrew Lydon scholarship, and the Iris O’Brien Foundation.
The authors have no conflicts of interest to declare. FC and EK are serving as one of the Guest Editors of this journal. We declare that FC and EK had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to LP.